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Patent 2510950 Summary

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(12) Patent: (11) CA 2510950
(54) English Title: CINNAMIC ACID 4-HYDROXYLASE
(54) French Title: ACIDE CINNAMIQUE-4-HYDROXYLASE
Status: Granted
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/53 (2006.01)
  • A01H 1/04 (2006.01)
  • A01H 3/04 (2006.01)
  • C07H 21/00 (2006.01)
  • C07K 16/16 (2006.01)
  • C07K 16/40 (2006.01)
  • C12N 5/04 (2006.01)
  • C12N 9/04 (2006.01)
  • G01N 33/573 (2006.01)
  • A23L 1/29 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventors :
  • CANTLE, SANDRA (Canada)
  • NARAYANAN, KARTHIKEYAN (Canada)
  • WANG-PRUSKI, GEFU (Canada)
(73) Owners :
  • WANG-PRUSKI, GEFU (Canada)
  • NARAYANAN, KARTHIKEYAN (Canada)
  • BABIN, SANDRA ELIZABETH (Canada)
The common representative is: WANG-PRUSKI, GEFU
(71) Applicants :
  • HER MAJESTY THE QUEEN IN RIGHT OF THE PROVINCE OF NOVA SCOTIA, AS REPRESENTED BY THE NOVA SCOTIA AGRICULTURAL COLLEGE (NSAC) (Canada)
(74) Agent: BORDEN LADNER GERVAIS LLP
(74) Associate agent:
(45) Issued: 2011-03-22
(22) Filed Date: 2005-06-14
(41) Open to Public Inspection: 2006-12-14
Examination requested: 2007-06-12
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data: None

Abstracts

English Abstract

After-cooking darkening is a gray-black discoloration of the potato tuber, formed after cooking by the oxidation of an iron-chlorogenic acid complex. Cinnamic acid 4-hydroxylase (C4H) is a key enzyme involved in the biosynthesis of chlorogenic acid. The full-length c4h gene was cloned and sequenced from both genomic DNA and cDNA of Russet Burbank tuber tissue by PCR and 5' and 3' RACE. The gene expression levels of c4h were examined by Northern hybridization in potato cultivars varying in susceptibility to after-cooking darkening. Results suggest that there is a relationship between the levels of c4h gene expression and the degree of after-cooking darkening in potato tubers.


French Abstract

Le noircissement après cuisson est un changement de couleur du tubercule de pomme de terre se produisant après la cuisson par oxydation d'un complexe fer-acide chlorogénique. La cinnamate 4-hydroxylase (C4H) est une enzyme clé qui intervient dans la biosynthèse de l'acide chlorogénique. Le gène c4h a été cloné et séquencé sur toute la longueur à partir de l'ADN et de l'ADNc du génome de tissus de tubercules de la variété Russet Burbank, cela par PCR et par RACE 5' et 3'. Le degré d'expression du gène de la c4h a été examiné par hybridation de Northern chez des cultivars de pommes de terre plus ou moins sensibles au noircissement après cuisson. Les résultats semblent indiquer l'existence d'un lien entre le degré d'expression du gène de la c4h et le degré de noircissement après cuisson des tubercules de pommes de terre.

Claims

Note: Claims are shown in the official language in which they were submitted.





121



WE CLAIM:


1. A purified and isolated nucleic molecule encoding a cinnamic acid 4-
hydroxylase (C4H) protein comprising SEQ ID NO:2.


2. The nucleic acid molecule according to claim 1 which comprises the sequence

shown in SEQ ID NO:1.


3. An isolated cinnamic acid 4-hydroxylase (C4H) protein comprising an amino
acid sequence shown in SEQ ID NO:2.


4. An antibody that specifically binds to the protein of claim 3.


5. An antisense oligonucleotide that is complimentary to a nucleic acid
sequence
according to claim 1 or 2.


6. A method of determining the susceptibility of a potato to after-cooking
darkening (ACD) comprising assaying a potato sample for (a) a nucleic acid
molecule
encoding a C4H protein shown in SEQ ID NO:2 or (b) a C4H protein shown in SEQ
ID
NO:2 wherein increased levels of the C4H nucleic acid or protein indicates
that the
potato is more susceptible to ACD.


7. The method of claim 6 wherein the sample is assayed for a nucleic acid
molecule encoding the C4H protein.


8. The method of claim 7 wherein the nucleic acid molecule has the sequence
shown in SEQ ID NO:1.


9. The method of claim 7 wherein the nucleic acid molecule is cDNA.


10. The method of claim 9 wherein the cDNA is detected using RT-PCR or real
time qualitative PCR.




122



11. The method of claim 10 wherein forward primer 5'-
GAAGGGAGAGATCAACGAGG-3' (SEQ ID NO:27) and reverse primer 5'-
FTCTGCACCAAACGTCC-3' (SEQ ID NO:28) are used in the method.


12. The method of claim 6 wherein the potato is the potato cultivar Russet
Burbank
or Shepody.

Description

Note: Descriptions are shown in the official language in which they were submitted.



DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME DE _2

NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.

JUMBO APPLICATIONS / PATENTS

THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

THIS IS VOLUME 1 OF 2

NOTE: For additional volumes please contact the Canadian Patent Office.


CA 02510950 2005-06-14

1

B&P File No: 14756-2
Title: Cinnamic Acid 4-Hydroxylase

FIELD OF THE INVENTION
The invention related to a novel cinnamic acid 4-hydroxylase gene and protein
from potato and uses thereof.

BACKGROUND OF THE INVENTION
After-cooking darkening (ACD) is a non-enzymatic gray-black discoloration
of potato tuber flesh occurring after cooking. The discoloration is due to the
formation of a colorless iron-chlorogenic acid complex during the cooking
process,
which upon exposure to air, oxidizes to form the dark ferridichlorogenic acid
(Dale
and Mackay, 1994). To prevent the discoloration caused by ACD, processors in
the
French fry industry treat the French fried potato strips with sodium acid
pyrophosphate (SAPP, Na2H2P7O7). Sodium acid pyrophosphate reduces darkening
by complexing with the iron in the tuber. In this capacity the iron is held in
a
nonionizable form and cannot take part in the reaction with chlorogenic acid
(Smith,
1987). A rise in the number of French fry processing industries has led to an
increase
in SAPP usage. The phosphorus residue released from SAPP during processing,
has
made it mandatory to eliminate SAPP from industrial wastewater. This currently
involves the removal of phosphorous from wastewater through chemical
precipitation,
adding further to processing costs for the French fry industry (Wang-Pruski
and
Nowak, 2004). Considering the millions of dollars per year that SAPP costs the
industry, it would be beneficial both from economical and environmental
standpoints
to reduce or eliminate the use of SAPP in the processing industry.
Traditional breeding has led to the development of many low-ACD cultivars,
including the cultivars Red Pontiac and Yukon Gold. However, cultivars for
French
fry production must also possess traits essential for processing, specifically
oblong
tuber shape, shallow eyes, high specific gravity, low reducing sugars, high
yield, and
resistance to diseases (Bradshaw et al., 1998). Currently, Russet Burbank and
Shepody are the primary cultivars used in the French fry processing industry
in
Canada. Both cultivars require the use of SAPP to prevent the darkening. To
date no
cultivars are available that possess all the traits essential for French fry
processing, as


CA 02510950 2005-06-14

2
well as complete resistance to ACD (Wang-Pruski, personal communication).
Chlorogenic acid (CA) is not only involved in ACD but it also has various
biological roles, including the involvement in defense mechanisms against
insects or
phytopathogens, disease and fungal resistance, growth regulation, and wound
response (Kiihnl et al., 1987; Yao et at., 1995; Friedman, 1997; Griffiths and
Bain,
1997). In potatoes specifically, CA is able to provide covalent cross-links
between
polysaccharides and cell well proteins; making the cell wall stronger and more
resistant to invading pathogens (Yao et al., 1995). Once the threat (pathogen
or
disease) subsides, normal oxidative processes lower the accumulated CA in
suberized
tissues (Friedman, 1997). Chlorogenic acid accounts for up to 90% of the total
phenolic compounds present in the potato tuber (Griffiths and Bain, 1997;
Lewis et
al., 1998; Lugasi et al., 1999; Percival and Baird, 2000). Approximately 50%
of the
CA is located in the potato peel and adjoining tissues. Chlorogenic acid is
synthesized via the phenylpropanoid pathway, which has not been explored in
great
detail, especially in species from the Solanaceae family.
Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11) is an essential enzyme for
the biosynthesis of CA and therefore is thought to play a key role in the ACD
mechanism. Cinnamic acid 4-hydroxylase catalyzes the hydroxylation of t-
cinnamic
acid to form p-coumaric acid, during the synthesis of CA. The C4H enzyme
belongs
to the CYP73 family of plant cytochrome P450 proteins. C4H enzymatic activity
is
induced by wounding, light, and pathogen infection in various plant species
(Tanaka
et al., 1974; Fahrendorf and Dixon, 1993; Bell-Lelong et al., 1997; Petersen,
2003).
Class I and II forms of the gene encoding C4H have been sequenced in many
plant
species, including Arabidopsis, Jerusalem artichoke, red pepper, pea, alfalfa,
and
species of Populus and Citrus. Class I c4h is the predominate form found in
almost all
plant species, whereas the divergent class II form has only been isolated from
orange
and French bean. The divergent class II c4h has approximately 60% sequence
similarity to the class I form and differs in the N-terminus and three
internal domains
(Betz et al., 2001; Blee et al., 2001).
The gene expression level of c4h depends on the specific plant species, tissue
type, as well as stress and environmental factors (Whitbred and Schuler,
2000). Bell-
Lelong et al. (1997) and Mizutani et al. (1997) found that in Arabidopsis, c4h
was


CA 02510950 2005-06-14

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expressed in all tissues analyzed including leaves, seedlings, stems, flowers,
and
roots. The higher levels were found in the stems and roots, possibly because
of
C4H's role in the production of the monolignols required for lignification.
The c4h
gene has not been sequenced nor has its expression profile been identified in
potato.
To date, no genes in potato have been identified that relate to the control of
ACD.
SUMMARY OF THE INVENTION
The present invention relates to a nucleic acid sequence that encodes cinnamic
acid 4-hydroxylase (C4H) from potato. The full length genomic DNA and cDNA of
the gene for the enzyme are identified. Further, its gene function at gene
expression
levels of this enzyme in potato has been confirmed to affect the chlorogenic
acid
biosynthesis in potato tubers. Its expression level is also correlated with
the degree of
the darkness in potatoes, the negative trait of after-cooking darkening (ACD)
affecting the quality of table stock and processing varieties.
Accordingly, the present invention provides an isolated nucleic acid molecule
comprising a sequence encoding the C4H enzyme. The invention also includes the
corresponding polypeptide, C4H.

In one embodiment, the purified and isolated nucleic acid molecule comprises
(a) a nucleic acid sequence encoding a protein as shown in Table 4 (SEQ ID
No.2);

(b) a nucleic acid sequence complementary to (a);

(c) a nucleic acid sequence that has substantial homology to (a) or (b);

(d) a nucleic acid sequence that is an analog to a nucleic acid sequence of
(a),
(b), or (c);

(e) a fragment of (a) to (d) that is at least 15 bases, preferably 20 to 30
bases,
and which will hybridize to a nucleic acid sequence of (a), (b), (c) or (d)
under
stringent hybridization conditions; or

(f) a nucleic acid molecule differing from any of the nucleic acids of (a) to
(c)
in codon sequences due to the degeneracy of the genetic code.


CA 02510950 2005-06-14

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In a specific embodiment of the invention, an isolated nucleic acid molecule
is
provided having a sequence as shown in Table 3 (SEQ ID No. 1) or a fragment or
variant thereof.

The present invention also includes the isolated C4H protein of the invention.
In a preferred embodiment, the C4H protein has the amino acid sequence shown
in
Table 4 (SEQ ID. NO. 2) or a fragment or variant thereof.
The present invention also includes methods of modulating C4H gene or
protein expression or activity comprising administering a modulator of the C4H
gene
or protein to a cell or plant in need thereof.
In one embodiment, the present invention provides a method of enhancing
C4H gene expression comprising administering an effective amount an agent that
can
enhance the expression or activity of the C4H gene or protein. Methods of
enhancing
the C4H gene expression can be used in enhancing disease resistance to
pathogens as
well as enhancing the nutritional value of foods.
In another embodiment, the present invention provides a method of decreasing
C4H gene expression or activity comprising administering an effective amount
of a
C4H inhibitor to a cell or animal in need thereof. Methods of inhibiting C4H
gene
expression or C4H protein activity can be useful in reducing after-cooking
darkening
of food.
Other features and advantages of the present invention will become apparent
from the following detailed description. It should be understood, however,
that the
detailed description and the specific examples while indicating preferred
embodiments of the invention are given by way of illustration only, since
various
changes and modifications within the spirit and scope of the invention will
become
apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Northern hybridization analysis of c4h expression in Russet Burbank
(RB),
Russet Norkotah (RN), light-ACD diploid (Lt), and dark-ACD diploid (Dk). A.
Hybridization signals from one of the three replicates using the 472 bp c4h
probe.


CA 02510950 2005-06-14

Lane C is the transcribed c4h probe used as a positive control (710 nt in
length). B.
The corresponding total RNA separated by formaldehyde agarose (0.7%) gel
electrophoresis.

5 Figure 2. Relationship between c4h gene expression and ACD level in potato
tubers.
Black bars represent the mean relative intensity of c4h expression (left axis)
and gray
bars represent the digital measurement of ACD in the potato tubers (right
axis). A
lower pixel density reading (right axis) represents higher ACD. Different
upper case
letters represent significance for the mean relative intensity of c4h
expression,
according to Tukey's hsd test at - = 0.05. Different lower case letters
represent
significance between the ACD levels, according to Tukey's hsd test at 0.05.
Figure 3. Comparison of exon and intron lengths of the potato c4h gene to that
of
other plant species. Diagram not to scale.
Figure 4. Tissue sampling for HPLC analysis and iron content measurement and
RNA
extraction

Figure 5. Schematic representation of the methodology followed to achieve the
objectives in Example 2.

Figure 6. Graph showing the pattern of ACD distribution among 129 clones of
family
13610-T.

Figure 7. Graph showing the pattern of ACD distribution among 43 clones of
family
13395-B.

Figure 8. Total RNA isolation after DNAse treatment and phenol chloroform
extraction.
Figure 9. Single- stranded cDNA synthesis from isolated total RNA using AMV
reverse transcriptase and random primers.


CA 02510950 2005-06-14

6
Figure 10. Intensities of PCR products amplified after 27 cycles with varying
initial
copies.

Figure 11. The linear range of the PCT cycle showing sensitivity of the
imaging
device based on the gel picture shown above.

Figure 12. Gel electrophoresis showing increasing intensities of the PCR
products at
different PCR amplification cycles.

Figure 13. Gel electrophoresis showing C4H gene expression profiles along with
internal standard in dark and light clones of family 13610-T. PCR 1 to PCR 4
are the
four individually repeated PCR experiments.

Figure 14. Normalized gene expression level of four repeated PCR experiments
for
C4H gene in ACD dark and light clones of family 13610 - T.

Figure 15. Gel electrophoresis showing C4H gene expression profiles along with
internal standard in dark and light clones of family 13395-B. PCR 1 to PCR 4
are the
four individually repeated PCR experiments.
Figure 16. Normalized gene expression level of four repeated PCR experiments
for
C4H gene in ACD dark and light clones of family 13395 - B.

Figure 17. Gel electrophoresis showing C4H gene expression profiles along with
internal standard in cultivars Shepody and Russet Burbank.

Figure 18. Normalized gene expression level of four repeated PCR experiments
for
C4H gene in cultivar Shepody and Russet Burbank.

Figure 19: Fold changes in the expression of candidate genes in dark clones to
that of
the light clones in family 13610-T. The numbers above the bars are the mean
fold
changes for the C4H gene. The error bars are the standard error mean
calculated using
students t test.


CA 02510950 2005-06-14
7

Figure 20: Mean normalized C4H gene expression in ACD dark and light clones of
the diploid families 13610 - T and 13395 - B. The significances were
statistically
analyzed using one-way ANOVA at p < 0.05

Figure 21: Mean normalized C4H gene expression in Shepody and Russet Burbank.
The significances were statistically analyzed using one-way ANOVA at p < 0.05
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a DNA sequence that encodes cinnamic acid
4-hydroxylase (C4H) from potato. The full length genomic DNA and cDNA of the
gene for the enzyme are identified. Further, its gene function at gene
expression levels
of this enzyme in potato has been confirmed to affect the chlorogenic acid
biosynthesis in potato tubers. Its expression level is also correlated with
the degree of

the darkness in potatoes, the negative trait named as after-cooking darkening
(ACD)
affecting the quality of table stock and processing varieties.

I. NUCLEIC ACID MOLECULES OF THE INVENTION

As hereinbefore mentioned, the present invention relates to isolated C4H
nucleic acid molecules. The term "isolated" refers to a nucleic acid
substantially free
of cellular material or culture medium when produced by recombinant DNA
techniques, or chemical precursors, or other chemicals when chemically
synthesized.

The term "nucleic acid" is intended to include DNA and RNA and can be
either double stranded or single stranded. The term is also intended to
include a strand
that is a mixture of nucleic acid molecules and nucleic acid analogs and/or
nucleotide
analogs, or that is made entirely of nucleic acid analogs and/or nucleotide
analogs.
Broadly stated, the present invention provides an isolated nucleic acid
molecule encoding the C4H protein. Accordingly, the present invention provides
an
isolated nucleic acid molecule containing a sequence encoding C4H shown in
Table 4
or a fragment, variant, or analog thereof.


CA 02510950 2005-06-14

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In one embodiment, the purified and isolated nucleic acid molecule comprises
(a) a nucleic acid sequence encoding a C4H protein as shown in Table 4 (SEQ
ID No. 2);

(b) a nucleic acid sequence complementary to (a);

(c) a nucleic acid sequence that has substantial homology to (a) or (b);

(d) a nucleic acid sequence that is an analog to a nucleic acid sequence of
(a),
(b), or (c);

(e) a fragment of (a) to (d) that is at least 15 bases, preferably 20 to 30
bases,
and which will hybridize to a nucleic acid sequence of (a), (b), (c) or (d)
under
stringent hybridization conditions; or

(f) a nucleic acid molecule differing from any of the nucleic acids of (a) to
(c)
in codon sequences due to the degeneracy of the genetic code.

In a specific embodiment of the invention, the isolated nucleic acid molecule
has a sequence as shown in Table 3 (SEQ ID No. 1) or a fragment or variant
thereof.
The term "C4H" means cinnamic acid 4-hydroxylase and includes the nucleic
acid sequence as shown in Table 3 (SEQ ID No. 1) or the protein having the
amino
acid sequence shown in Table 4 (SEQ ID No. 2) as well as mutations, variants
and
fragments thereof that can catalyze the hydroxylation of t-cinnamic acid to p-
coumaric acid during the synthesis of cinnamic acid.

It will be appreciated that the invention includes nucleic acid molecules
encoding truncations of the C4H proteins of the invention, and analogs and
homologs
of the C4H proteins of the invention and truncations thereof, as described
below.

Further, it will be appreciated that the invention includes nucleic acid
molecules comprising nucleic acid sequences having substantial sequence
homology
with the nucleic acid sequences of the invention and fragments thereof. The
term
"sequences having substantial sequence homology" means those nucleic acid
sequences which have slight or inconsequential sequence variations from these
sequences, i.e. the sequences function in substantially the same manner to
produce
functionally equivalent proteins. The variations may be attributable to local
mutations


CA 02510950 2005-06-14

9
or structural modifications.

Generally, nucleic acid sequences having substantial homology include
nucleic acid sequences having at least 70%, preferably 80-90% identity with
the
nucleic acid sequences of the invention.

Sequence identity is most preferably assessed by the algorithm of the BLAST
version 2.1 program advanced search (BLAST is a series of programs that are
available online at http://www.ncbi.nlm.nih.gov/BLAST. The advanced blast
search
(http://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=1) is set to default
parameters. (ie
Matrix BLOSUM62; Gap existence cost 11; Per residue gap cost 1; Lambda ratio
0.85 default).). For example, if a nucleotide sequence (called "Sequence A")
has 90%
identity to a portion of the nucleotide sequence in Table 3, then Sequence A
will be
identical to the referenced portion of the nucleotide sequence in Table 3,
except that
Sequence A may include up to 10 point mutations, such as substitutions with
other
nucleotides, per each 100 nucleotides of the referenced portion of the
nucleotide
sequence in Table 3. Nucleotide sequences functionally equivalent to the C4H
transcript can occur in a variety of forms as described below.

The term "a nucleic acid sequence which is an analog" means a nucleic acid
sequence which has been modified as compared to the sequence of (a), (b) or
(c)
wherein the modification does not alter the utility of the sequence as
described herein.
The modified sequence or analog may have improved properties over the sequence
shown in (a), (b) or (c). One example of a modification to prepare an analog
is to
replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine
or
thymidine) of the sequence shown in Table I, with a modified base such as
xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-
halo
uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine,
pseudo uracil,
4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl
adenines,
8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino
guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other
8-
substituted guanines, other aza and deaza uracils, thymidines, cytosines,
adenines, or
guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.


CA 02510950 2005-06-14

Another example of a modification is to include modified phosphorous or
oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl
intersugar linkages or short chain heteroatomic or heterocyclic intersugar
linkages in
the nucleic acid molecule shown in Table 3. For example, the nucleic acid
sequences
5 may contain phosphorothioates, phosphotriesters, methyl phosphonates, and
phosphorodithioates.
A further example of an analog of a nucleic acid molecule of the invention is
a
peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate
backbone
in the DNA (or RNA), is replaced with a polyamide backbone which is similar to
that
10 found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA
analogs have
been shown to be resistant to degradation by enzymes and to have extended
lives in
vivo and in vitro. PNAs also bind stronger to a complementary DNA sequence due
to
the lack of charge repulsion between the PNA strand and the DNA strand. Other
nucleic acid analogs may contain nucleotides containing polymer backbones,
cyclic
backbones, or acyclic backbones. For example, the nucleotides may have
morpholino
backbone structures (U.S. Pat. No. 5,034,506).

Another aspect of the invention provides a nucleic acid molecule, and
fragments thereof having at least 15 bases, which hybridizes to the nucleic
acid
molecules of the invention under hybridization conditions. Such nucleic acid
molecules preferably hybridize to all or a portion of C4H or its complement
under
stringent conditions as defined herein (see Sambrook et al. (most recent
edition)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.; Ausubel et al. (eds.), 1995, Current Protocols in
Molecular
Biology, (John Wiley & Sons, NY)). The portion of the hybridizing nucleic
acids is
typically at least 15 (e.g. 20, 25, 30 or 50) nucleotides in length. The
hybridizing
portion of the hybridizing nucleic acid is at least 80% e.g. at least 95% or
at least 98%
identical to the sequence or a portion or all of a nucleic acid encoding a C4H
polypeptide, or its complement. Hybridizing nucleic acids of the type
described
herein can be used, for example, as a cloning probe, a primer (e.g. a PCR
primer) or a
diagnostic probe. Hybridization of the oligonucleotide probe to a nucleic acid
sample
typically is performed under stringent conditions. Nucleic acid duplex or
hybrid


CA 02510950 2005-06-14

11
stability is expressed as the melting temperature or Tm, which is the
temperature at
which a probe dissociates from a target DNA. This melting temperature is used
to
define the required stringency conditions. If sequences are to be identified
that are
related and substantially identical to the probe, rather than identical, then
it is useful
to first establish the lowest temperature at which only homologous
hybridization
occurs with a particular concentration of salt (e.g. SSC or SSPE). Then,
assuming
that 1% mismatching results in a 1 degree Celsius decrease in the Tm, the
temperature
of the final wash in the hybridization reaction is reduced accordingly (for
example, if
sequences having greater than 95% identity with the probe are sought, the
final wash

temperature is decreased by 5 degrees Celsius). In practice, the change in Tm
can be
between 0.5 degrees Celsius and 1.5 degrees Celsius per 1% mismatch. Low
stringency conditions involve hybridizing at about: 1XSSC, 0.1% SDS at 50 C.
High
stringency conditions are: 0.1XSSC, 0.1% SDS at 65 C. Moderate stringency is
about 1X SSC 0.1% SDS at 60 degrees Celsius. The parameters of salt
concentration
and temperature can be varied to achieve the optimal level of identity between
the
probe and the target nucleic acid.

Isolated and purified nucleic acid molecules having sequences which differ
from the nucleic acid sequence shown in Table 3 due to degeneracy in the
genetic
code are also within the scope of the invention. The genetic code is
degenerate so
other nucleic acid molecules, which encode a polypeptide identical to the C4H
amino
acid sequence (Table 4) may also be used.

Nucleic acid molecules from C4H can be isolated by preparing a labelled
nucleic acid probe based on all or part of the nucleic acid sequences as shown
in
Table 3, and using this labelled nucleic acid probe to screen an appropriate
DNA
library (e.g. a cDNA or genomic DNA library). Nucleic acids isolated by
screening
of a cDNA or genomic DNA library can be sequenced by standard techniques.
Another method involves comparing the C4H sequence to other sequences, for
example using bioinformatics techniques such as database searches or alignment
strategies, and detecting the presence of a C4H nucleic acid sequence.

Nucleic acid molecules of the invention can also be isolated by selectively
amplifying a nucleic acid using the polymerase chain reaction (PCR) methods
and


CA 02510950 2005-06-14

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cDNA or genomic DNA. It is possible to design synthetic oligonucleotide
primers
from the nucleic acid molecules as shown in Table 3 for use in PCR. A nucleic
acid
can be amplified from cDNA or genomic DNA using these oligonucleotide primers
and standard PCR amplification techniques. The nucleic acid so amplified can
be
cloned into an appropriate vector and characterized by DNA sequence analysis.
It
will be appreciated that cDNA may be prepared from mRNA, by isolating total
cellular mRNA by a variety of techniques, for example, by using the
guanidinium-
thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-
5299
(1979). cDNA is then synthesized from the mRNA using reverse transcriptase
(for
example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda,
MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St.
Petersburg, FL).

An isolated nucleic acid molecule of the invention which is RNA can be
isolated by cloning a cDNA encoding a novel protein of the invention into an
appropriate vector which allows for transcription of the cDNA to produce an
RNA
molecule which encodes the C4H protein. For example, a cDNA can be cloned
downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA
can
be transcribed in vitro with T7 polymerase, and the resultant RNA can be
isolated by
standard techniques.

A nucleic acid molecule of the invention may also be chemically synthesized
using standard techniques. Various methods of chemically synthesizing
polydeoxynucleotides are known, including solid-phase synthesis which, like
peptide
synthesis, has been fully automated in commercially available DNA synthesizers
(See
e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent
No.
4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).

The sequence of a nucleic acid molecule of the invention may be inverted
relative to its normal presentation for transcription to produce an antisense
nucleic
acid molecule. Preferably, an antisense sequence is constructed by inverting a
region
preceding the initiation codon or an unconserved region. In particular, the
nucleic
acid sequences contained in the nucleic acid molecules of the invention or a
fragment
thereof, preferably a nucleic acid sequence shown in Table 3 may be inverted
relative


CA 02510950 2005-06-14

13
to its normal presentation for transcription to produce antisense nucleic acid
molecules.

The antisense nucleic acid molecules of the invention or a fragment thereof,
may be chemically synthesized using naturally occurring nucleotides or
variously
modified nucleotides designed to increase the biological stability of the
molecules or
to increase the physical stability of the duplex formed with mRNA or the
native gene
e.g. phosphorothioate derivatives and acridine substituted nucleotides. The
antisense
sequences may be produced biologically using an expression vector introduced
into
cells in the form of a recombinant plasmid, phagemid or attenuated virus in
which
antisense sequences are produced under the control of a high efficiency
regulatory
region, the activity of which may be determined by the cell type into which
the vector
is introduced.

The invention also provides nucleic acids encoding fusion proteins comprising
a novel protein of the invention and a selected protein, or a selectable
marker protein
(see below).

H. NOVEL PROTEINS OF THE INVENTION

The invention further includes an isolated C4H protein encoded by the nucleic
acid molecules of the invention. Within the context of the present invention,
a protein
of the invention may include various structural forms of the primary protein
which
retain biological activity.

Broadly stated, the present invention provides an isolated C4H protein from
potatoes.

In a preferred embodiment of the invention, the C4H protein has the amino
acid sequence as shown in Table 4 (SEQ ID No. 2) or a fragment or variant
thereof.
In addition to full length amino acid sequences, the proteins of the present
invention also include truncations of the protein, and analogs, and homologs
of the
protein and truncations thereof as described herein. Truncated proteins may
comprise
peptides of at least fifteen amino acid residues.

Analogs or variants of the protein having the amino acid sequence shown in


CA 02510950 2005-06-14

14
Table 4 and/or truncations thereof as described herein, may include, but are
not
limited to an amino acid sequence containing one or more amino acid
substitutions,
insertions, and/or deletions. Amino acid substitutions may be of a conserved
or non-
conserved nature. Conserved amino acid substitutions involve replacing one or
more
amino acids of the proteins of the invention with amino acids of similar
charge, size,
and/or hydrophobicity characteristics. When only conserved substitutions are
made
the resulting analog should be functionally equivalent. Non-conserved
substitutions
involve replacing one or more amino acids of the amino acid sequence with one
or
more amino acids which possess dissimilar charge, size, and/or hydrophobicity
characteristics.

One or more amino acid insertions may be introduced into the amino acid
sequence shown in Table 4. Amino acid insertions may consist of single amino
acid
residues or sequential amino acids ranging from 2 to 15 amino acids in length.
For
example, amino acid insertions may be used to destroy target sequences so that
the
protein is no longer active. This procedure may be used in vivo to inhibit the
activity
of a protein of the invention.

Deletions may consist of the removal of one or more amino acids, or discrete
portions from the amino acid sequence shown in Table 4. The deleted amino
acids
may or may not be contiguous. The lower limit length of the resulting analog
with a
deletion mutation is about 10 amino acids, preferably 100 amino acids.

Analogs of a protein of the invention may be prepared by introducing
mutations in the nucleotide sequence encoding the protein. Mutations in
nucleotide
sequences constructed for expression of analogs of a protein of the invention
must
preserve the reading frame of the coding sequences. Furthermore, the mutations
will
preferably not create complementary regions that could hybridize to produce
secondary mRNA structures, such as loops or hairpins, which could adversely
affect
translation of the receptor mRNA.

Mutations may be introduced at particular loci by synthesizing
oligonucleotides containing a mutant sequence, flanked by restriction sites
enabling
ligation to fragments of the native sequence. Following ligation, the
resulting


CA 02510950 2005-06-14

reconstructed sequence encodes an analog having the desired amino acid
insertion,
substitution, or deletion.

Alternatively, oligonucleotide-directed site specific mutagenesis procedures
may be employed to provide an altered gene having particular codons altered
5 according to the substitution, deletion, or insertion required. Deletion or
truncation of
a protein of the invention may also be constructed by utilizing convenient
restriction
endonuclease sites adjacent to the desired deletion. Subsequent to
restriction,
overhangs may be filled in, and the DNA religated. Exemplary methods of making
the alterations set forth above are disclosed by Sambrook et al (Molecular
Cloning: A
10 Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989).

A homologous protein includes a protein with an amino acid sequence having
at least 70%, preferably 80-90%, most preferably 95% identity with the amino
acid
sequence as shown in Table 4. As with the nucleic acid molecules of the
invention,
identity is calculated according to methods known in the art. Sequence
identity is
15 most preferably assessed by the algorithm of BLAST version 2.1 advanced
search.
BLAST is a series of programs that are available online at
http://www.ncbi.nlm.nih.gov/BLAST. The advanced blast search
(http:://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=l) is set to default
parameters
(i.e. Matrix BLOSUM62, Gap existence cost 11; Per residue gap cost 1; Lambda
ration 0.85 default).

The invention also contemplates isoforms of the proteins of the invention. An
isoform contains the same number and kinds of amino acids as a protein of the
invention, but the isoform has a different molecular structure. The isoforms
contemplated by the present invention are those having the same properties as
a
protein of the invention as described herein.

The present invention also includes a protein of the invention conjugated with
a selected protein, or a selectable marker protein (see below) to produce
fusion
proteins. Additionally, immunogenic portions of a protein of the invention are
within
the scope of the invention.

The proteins of the invention (including truncations, analogs, etc.) may be


CA 02510950 2005-06-14

16
prepared using recombinant DNA methods. Accordingly, the nucleic acid
molecules
of the present invention having a sequence which encodes a protein of the
invention
may be incorporated in a known manner into an appropriate expression vector
which
ensures good expression of the protein. Possible expression vectors include
but are
not limited to cosmids, plasmids, or modified viruses (e.g. replication
defective
retroviruses, adenoviruses and adeno-associated viruses), so long as the
vector is
compatible with the host cell used. The expression vectors are "suitable for
transformation of a host cell", means that the expression vectors contain a
nucleic
acid molecule of the invention and regulatory sequences selected on the basis
of the
host cells to be used for expression, which is operatively linked to the
nucleic acid
molecule. Operatively linked is intended to mean that the nucleic acid is
linked to
regulatory sequences in a manner which allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector of the
invention containing a nucleic acid molecule of the invention, or a fragment
thereof,
and the necessary regulatory sequences for the transcription and translation
of the
inserted protein-sequence. Suitable regulatory sequences may be derived from a
variety of sources, including bacterial, fungal, or viral genes (For example,
see the
regulatory sequences described in Goeddel, Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of
appropriate regulatory sequences is dependent on the host cell chosen, and may
be
readily accomplished by one of ordinary skill in the art. Examples of such
regulatory
sequences include: a transcriptional promoter and enhancer or RNA polymerase
binding sequence, a ribosomal binding sequence, including a translation
initiation
signal. Additionally, depending on the host cell chosen and the vector
employed,
other sequences, such as an origin of replication, additional DNA restriction
sites,
enhancers, and sequences conferring inducibility of transcription may be
incorporated
into the expression vector. It will also be appreciated that the necessary
regulatory
sequences may be supplied by the native protein and/or its flanking regions.

The invention further provides a recombinant expression vector comprising a
DNA nucleic acid molecule of the invention cloned into the expression vector
in an
antisense orientation. That is, the DNA molecule is operatively linked to a
regulatory


CA 02510950 2005-06-14

17
sequence in a manner which allows for expression, by transcription of the DNA
molecule, of an RNA molecule which is antisense to a nucleotide sequence
comprising the nucleotides as shown in Table 3. Regulatory sequences
operatively
linked to the antisense nucleic acid can be chosen which direct the continuous
expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain a
selectable marker gene which facilitates the selection of host cells
transformed or
transfected with a recombinant molecule of the invention. Examples of
selectable
marker genes are genes encoding a protein such as G418 and hygromycin which
confer resistance to certain drugs, B-galactosidase, chloramphenicol
acetyltransferase,
or firefly luciferase . Transcription of the selectable marker gene is
monitored by
changes in the concentration of the selectable marker protein such as B-
galactosidase,
chloramphenicol acetyltransferase, or firefly luciferase. If the selectable
marker gene
encodes a protein conferring antibiotic resistance such as neomycin resistance
transformant cells can be selected with G418. Cells that have incorporated the
selectable marker gene will survive, while the other cells die. This makes it
possible
to visualize and assay for expression of recombinant expression vectors of the
invention and in particular to determine the effect of a mutation on
expression and
phenotype. It will be appreciated that selectable markers can be introduced on
a
separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode a
fusion moiety which provides increased expression of the recombinant protein;
increased solubility of the recombinant protein; and aid in the purification
of a target
recombinant protein by acting as a ligand in affinity purification. For
example, a
proteolytic cleavage site may be added to the target recombinant protein to
allow
separation of the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein.

Recombinant expression vectors can be introduced into host cells to produce a
transformed host cell. The term "transformant host cell" is intended to
include
prokaryotic and eukaryotic cells which have been transformed or transfected
with a
recombinant expression vector of the invention. The terms "transformed with",


CA 02510950 2005-06-14

18
"transfected with", "transformation" and "transfection" are intended to
encompass
introduction of nucleic acid (e.g. a vector) into a cell by one of many
possible
techniques known in the art. Prokaryotic cells can be transformed with nucleic
acid
by, for example, electroporation or calcium-chloride mediated transformation.
Nucleic acid can be introduced into mammalian cells via conventional
techniques
such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-
mediated transfection, lipofectin, electroporation or microinjection. Suitable
methods
for transforming and transfecting host cells can be found in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory press (1989)), and other laboratory textbooks.

Methodologies to introduce plant recombinant expression vectors into a plant
cell, also referred to herein as "transformation", are well known to the art
and
typically vary depending on the plant cell that is selected. General
techniques to
introduce recombinant expression vectors in cells include, electroporation;
chemically
mediated techniques, for example CaCl2 mediated nucleic acid uptake; particle
bombardment (biolistics); the use of naturally infective nucleic acid
sequences, for
example virally derived nucleic acid sequences, or Agrobacterium or Rhizobium
derived sequences, polyethylene glycol (PEG) mediated nucleic acid uptake,
microinjection and the use of silicone carbide whiskers.
In preferred embodiments, a transformation methodology is selected which
will allow the integration of the C4H nucleic acid sequence in the plant
cell's
genome, and preferably the plant cell's nuclear genome. In accordance herewith
this
is considered particularly desirable as the use of such a methodology will
result in the
transfer of the C4H nucleic acid sequence to progeny plants upon sexual
reproduction. Transformation methods that may be used in this regard include
biolistics and Agrobacterium mediated methods.
Transformation methodologies for dicotyledenous plant species are
well known. Generally, Agrobacterium mediated transformation is used because
of its
high efficiency, as well as the general susceptibility by many, if not all,
dicotyledenous plant species. Agrobacterium transformation generally involves
the
transfer of a binary vector (e.g. pGreenII0129), comprising the nucleic acid
sequence


CA 02510950 2005-06-14

19
of the present invention from E. coli to a suitable Agrobacterium strain (e.g.
GV3101,
EHA101 and LBA4404) by, for example, tri-parental mating with an E. coli
strain
carrying the recombinant binary vector and an E. coli strain carrying a helper
plasmid
capable of mobilizing the binary vector to the target Agrobacterium strain, or
by DNA
transformation of the Agrobacterium strain (Hofgen et al., Nucl. Acids. Res.,
1988,
16:9877). Other techniques that may be used to transform dicotyledenous plant
cells
include biolistics (Sanford, 1988, Trends in Biotechn. 6:299-302);
electroporation
(Fromm et al., 1985, Proc. Natl. Acad. Sci. USA., 82:5824-5828); PEG mediated
DNA uptake (Potrykus et al., 1985, Mol. Gen. Genetics, 199:169-177);
microinjection

(Reich et al., Bio/Techn., 1986, 4:1001-1004); and silicone carbide whiskers
(Kaeppler et al., 1990, Plant Cell Rep., 9:415-418) or in planta
transformation using,
for example, a flower dipping methodology (Clough and Bent, 1998, Plant J.,
16:735-
743).
Monocotyledonous plant species may be transformed using a variety
of methodologies including particle bombardment (Christou et al., 1991,
Biotechn.
9:957-962; Weeks et al., Plant Physiol., 1993, 102:1077-1084; Gordon-Kamm et
al.,
Plant Cell, 1990, 2:5603-618); PEG mediated DNA uptake (European Patents 0292
435; 0392 225) or Agrobacterium mediated transformation (Goto-Fumiyuki et al.,
1999, Nature-Biotech. 17:282-286).
The exact plant transformation methodology may vary somewhat
depending on the plant species and the plant cell type (e.g. seedling derived
cell types
such as hypocotyls and cotyledons or embryonic tissue) that is selected as the
cell
target for transformation. As hereinbefore mentioned in a particularly
preferred
embodiment potato is used. A methodology to obtain potato transformants is
available
(De Block M. 1988. Genotype-independent leaf disc transformation of potato
(Solanum tuberosum) using Agrobacterium tumefaciens. Theor Appl Genet 76: 767-
774)
Following transformation, the plant cells are grown and upon the
emergence of differentiating tissue, such as shoots and roots, mature plants
are
regenerated. Typically a plurality of plants is regenerated. Methodologies to
regenerate plants are generally plant species and cell type dependent and will
be
known to those skilled in the art. Further guidance with respect to plant
tissue culture


CA 02510950 2005-06-14

may be found in, for example: Plant Cell and Tissue Culture, 1994, Vasil and
Thorpe
Eds., Kluwer Academic Publishers; and in: Plant Cell Culture Protocols
(Methods in
Molecular Biology 111), 1999, Hall Eds, Humana Press.
The proteins of the invention may also be prepared by chemical synthesis
5 using techniques well known in the chemistry of proteins such as solid phase
synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in
homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E.
Wansch, Vol. 15 I and II, Thieme, Stuttgart).

III. APPLICATIONS

10 The present invention includes all uses of the nucleic acid molecule and
C4H
proteins of the invention including, but not limited to, the preparation of
antibodies
and antisense oligonucleotides, the preparation of diagnostic assays, the
isolation of
substances that modulate C4H expression and/or activity as well as the use of
the C4H
nucleic acid sequences and proteins and modulators thereof. Some of the uses
are
15 further described below.

(a) Antibodies
The isolation of the C4H protein enables the preparation of antibodies
specific
for C4H. Accordingly, the present invention provides an antibody that binds to
a C4H
protein.
20 Conventional methods can be used to prepare the antibodies. For example, by
using a peptide of C4H, polyclonal antisera or monoclonal antibodies can be
made
using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be
immunized with an immunogenic form of the peptide which elicits an antibody
response in the mammal. Techniques for conferring immunogenicity on a peptide
include conjugation to carriers or other techniques well known in the art. For
example, the protein or peptide can be administered in the presence of
adjuvant. The
progress of immunization can be monitored by detection of antibody titers in
plasma
or serum. Standard ELISA or other immunoassay procedures can be used with the
immunogen as antigen to assess the levels of antibodies. Following
immunization,
antisera can be obtained and, if desired, polyclonal antibodies isolated from
the sera.


CA 02510950 2005-06-14

21
To produce monoclonal antibodies, antibody producing cells (lymphocytes)
can be harvested from an immunized animal and fused with myeloma cells by
standard somatic cell fusion procedures thus immortalizing these cells and
yielding
hybridoma cells. Such techniques are well known in the art, (e.g., the
hybridoma
technique originally developed by Kohler and Milstein (Nature 256, 495-497
(1975))
as well as other techniques such as the human B-cell hybridoma technique
(Kozbor et
al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce
human
monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy
(1985)
Allen R. Bliss, Inc., pages 77-96), and screening of combinatorial antibody
libraries
(Huse et al., Science 246, 1275 (1989)). Hybridoma cells can be screened
immunochemically for production of antibodies specifically reactive with the
peptide
and the monoclonal antibodies can be isolated. Therefore, the invention also
contemplates hybridoma cells secreting monoclonal antibodies with specificity
for
C4H as described herein.
The term "antibody" as used herein is intended to include fragments thereof
which also specifically react with C4H. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility in the same
manner as
described above. For example, F(ab')2 fragments can be generated by treating
antibody with pepsin. The resulting F(ab')2 fragment can be further treated to
produce
Fab' fragments.
Chimeric antibody derivatives, i.e., antibody molecules that combine a non-
human animal variable region and a human constant region are also contemplated
within the scope of the invention. Chimeric antibody molecules can include,
for
example, the antigen binding domain from an antibody of a mouse, rat, or other
species, with human constant regions. Conventional methods may be used to make
chimeric antibodies containing the immunoglobulin variable region which
recognizes
the gene product of C4H antigens of the invention (See, for example, Morrison
et at.,
Proc. Nat! Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452
(1985),
Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No.
4,816,397;
Tanaguchi et al., European Patent Publication EP171496; European Patent
Publication
0173494, United Kingdom patent GB 2177096B). It is expected that chimeric


CA 02510950 2005-06-14

22
antibodies would be less immunogenic in a human subject than the corresponding
non-chimeric antibody.
Monoclonal or chimeric antibodies specifically reactive with a protein of the
invention as described herein can be further humanized by producing human
constant
region chimeras, in which parts of the variable regions, particularly the
conserved
framework regions of the antigen-binding domain, are of human origin and only
the
hypervariable regions are of non-human origin. Such immunoglobulin molecules
may
be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad.
Sci.
U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983);
Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication
W092/06193
or EP 0239400). Humanized antibodies can also be commercially produced
(Scotgen
Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
Specific antibodies, or antibody fragments, reactive against C4H proteins may
also be generated by screening expression libraries encoding immunoglobulin
genes,
or portions thereof, expressed in bacteria with peptides produced from the
nucleic acid
molecules of C4H. For example, complete Fab fragments, VH regions and FV
regions
can be expressed in bacteria using phage expression libraries (See for example
Ward
et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281
(1989); and
McCafferty et al. Nature 348, 552-554 (1990)). Alternatively, a SCID-hu mouse,
for
example the model developed by Genpharm, can be used to produce antibodies or
fragments thereof.
(b) Antisense Oligonucleotides
Isolation of a nucleic acid molecule encoding C4H enables the production of
antisense oligonucleotides that can modulate the expression and/or activity of
C4H.
Accordingly, the present invention provides an antisense oligonucleotide that
is
complementary to a nucleic acid sequence encoding C4H. In one embodiment, the
nucleic acid sequence is a shown in Table 3.
The term "antisense oligonucleotide" as used herein means a nucleotide
sequence that is complementary to its target.
The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or
nucleoside monomers consisting of naturally occurring bases, sugars, and
intersugar
(backbone) linkages. The term also includes modified or substituted oligomers


CA 02510950 2005-06-14

23
comprising non-naturally occurring monomers or portions thereof, which
function
similarly. Such modified or substituted oligonucleotides may be preferred over
naturally occurring forms because of properties such as enhanced cellular
uptake, or
increased stability in the presence of nucleases. The term also includes
chimeric
oligonucleotides which contain two or more chemically distinct regions. For
example,
chimeric oligonucleotides may contain at least one region of modified
nucleotides that
confer beneficial properties (e.g. increased nuclease resistance, increased
uptake into
cells), or two or more oligonucleotides of the invention may be joined to form
a
chimeric oligonucleotide.
The antisense oligonucleotides of the present invention may be ribonucleic or
deoxyribonucleic acids and may contain naturally occurring bases including
adenine,
guanine, cytosine, thymidine and uracil. The oligonucleotides may also contain
modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-
propyl
and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza
cytosine
and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-
aminoadenine, 8-
thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-
substituted
adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl
guanines, 8-
hydroxyl guanine and other 8-substituted guanines, other aza and deaza
uracils,
thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-
trifluoro
cytosine.
Other antisense oligonucleotides of the invention may contain modified
phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl
or
cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic
intersugar
linkages. For example, the antisense oligonucleotides may contain
phosphorothioates,
phosphotriesters, methyl phosphonates, and phosphorodithioates. In an
embodiment
of the invention there are phosphorothioate bonds links between the four to
six 3'-
terminus bases. In another embodiment phosphorothioate bonds link all the
nucleotides.
The antisense oligonucleotides of the invention may also comprise nucleotide
analogs that may be better suited as therapeutic or experimental reagents. An
example
of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the
deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced
with a


CA 02510950 2005-06-14

24
polyamide backbone which is similar to that found in peptides (P.E. Nielsen,
et al
Science 1991, 254, 1497). PNA analogues have been shown to be resistant to
degradation by enzymes and to have extended lives in vivo and in vitro. PNAs
also
bind stronger to a complementary DNA sequence due to the lack of charge
repulsion
between the PNA strand and the DNA strand. Other oligonucleotides may contain
nucleotides containing polymer backbones, cyclic backbones, or acyclic
backbones.
For example, the nucleotides may have morpholino backbone structures (U.S.
Pat. No.
5,034,506). Oligonucleotides may also contain groups such as reporter groups,
a
group for improving the pharmacokinetic properties of an oligonucleotide, or a
group
for improving the pharmacodynamic properties of an antisense oligonucleotide.
Antisense oligonucleotides may also have sugar mimetics.
The antisense nucleic acid molecules may be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known in the art.
The
antisense nucleic acid molecules of the invention or a fragment thereof, may
be
chemically synthesized using naturally occurring nucleotides or variously
modified
nucleotides designed to increase the biological stability of the molecules or
to increase
the physical stability of the duplex formed with mRNA or the native gene e.g.
phosphorothioate derivatives and acridine substituted nucleotides. The
antisense
sequences may be produced biologically using an expression vector introduced
into
cells in the form of a recombinant plasmid, phagemid or attenuated virus in
which
antisense sequences are produced under the control of a high efficiency
regulatory
region, the activity of which may be determined by the cell type into which
the vector
is introduced.
The antisense oligonucleotides may be introduced into tissues or cells using
techniques in the art including vectors (retroviral vectors, adenoviral
vectors and DNA
virus vectors) or physical techniques such as microinjection. The antisense
oligonucleotides may be directly administered in vivo or may be used to
transfect cells
in vitro which are then administered in vivo.


CA 02510950 2005-06-14

(c) Diagnostic Assays
The present inventors have determined that there is a correlation between
C4H and susceptibility to ACD, the higher the level of the C4H gene, the more
susceptible the plant is to ACD.
5 Accordingly, the present invention provides a method of determining the
susceptibility of a plant to ACD comprising assaying a sample for (a) a
nucleic acid
molecule encoding a C4H protein or a fragment thereof or (b) a C4H protein or
a
fragment thereof. The C4H protein preferably has the sequence shown in Table
4.
(i) Nucleic acid molecules
10 The nucleic acid molecules encoding C4H as described herein or fragments
thereof, allow those skilled in the art to construct nucleotide probes and
primers for
use in the detection of nucleotide sequences encoding C4H or fragments thereof
in
samples, preferably plant samples such as edible plants. Edible plants include
but are
not limited to, root vegetables and fruits. Examples of root vegetables
include
15 potatoes. Examples of fruit include apples and pears.
Accordingly, the present invention provides a method for detecting a nucleic
acid molecule encoding C4H in a sample comprising contacting the sample with a
nucleotide probe capable of hybridizing with the nucleic acid molecule to form
a
hybridization product, under conditions which permit the formation of the
20 hybridization product, and assaying for the hybridization product.
Example of probes that may be used in the above method include fragments of
the nucleic acid sequences shown in Table 3 or SEQ.ID.NO.: 1. A nucleotide
probe
may be labelled with a detectable substance such as a radioactive label which
provides for an adequate signal and has sufficient half-life such as 32P, 3H,
14C or the
25 like. Other detectable substances which may be used include antigens that
are
recognized by a specific labelled antibody, fluorescent compounds, enzymes,
antibodies specific for a labelled antigen, and chemiluminescence. An
appropriate
label may be selected having regard to the rate of hybridization and binding
of the
probe to the nucleic acid to be detected and the amount of nucleic acid
available for
hybridization. Labelled probes may be hybridized to nucleic acids on solid
supports
such as nitrocellulose filters or nylon membranes as generally described in
Sambrook
et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleotide


CA 02510950 2005-06-14

26
probes may be used to detect genes, preferably in plant cells, that hybridize
to the
nucleic acid molecule of the present invention preferably, nucleic acid
molecules
which hybridize to the nucleic acid molecule of the invention under stringent
hybridization conditions as described herein.

In one embodiment, the hybridization assay can be a Southern analysis where
the sample is tested for a DNA sequence that hybridizes with a C4H specific
probe.
In another embodiment, the hybridization assay can be a Northern analysis
where the
sample is tested for an RNA sequence that hybridizes with a C4H specific
probe.
Southern and Northern analyses may be performed using techniques known in the
art
(see for example, Current Protocols in Molecular Biology, Ausubel, F. et al.,
eds.,
John Wiley & Sons).
Nucleic acid molecules encoding a C4H protein can be selectively amplified
in a sample using the polymerase chain reaction (PCR) methods and cDNA or
genomic DNA. It is possible to design synthetic oligonucleotide primers from
the

nucleotide sequence shown in Table 3,(SEQ.ID.NO.:1) for use in PCR. A nucleic
acid can be amplified from cDNA or genomic DNA using oligonucleotide primers
and standard PCR amplification techniques. The amplified nucleic acid can be
cloned
into an appropriate vector and characterized by DNA sequence analysis. cDNA
may
be prepared from mRNA, by isolating total cellular mRNA by a variety of
techniques,
for example, by using the guanidinium-thiocyanate extraction procedure of
Chirgwin
et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the
mRNA using reverse transcriptase (for example, Moloney MLV reverse
transcriptase
available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available
from Seikagaku America, Inc., St. Petersburg, FL).
Samples may be screened using probes to detect the presence of a C4H gene
by a variety of techniques. Genomic DNA used for the diagnosis may be obtained
from cells. The DNA may be isolated and used directly for detection of a
specific
sequence or may be PCR amplified prior to analysis. RNA or cDNA may also be
used. To detect a specific DNA sequence hybridization using specific
oligonucleotides, direct DNA sequencing, restriction enzyme digest, RNase
protection, chemical cleavage, and ligase-mediated detection are all methods
which
can be utilized. Oligonucleotides specific to mutant sequences can be
chemically


CA 02510950 2005-06-14

27
synthesized and labelled radioactively with isotopes, or non-radioactively
using biotin
tags, and hybridized to individual DNA samples immobilized on membranes or
other
solid-supports by dot-blot or transfer from gels after electrophoresis. The
presence or
absence of the C4H sequences is then visualized using methods such as
autoradiography, fluorometry, or colorimetric reaction

Direct DNA sequencing reveals the presence of C4H DNA. Cloned DNA
segments may be used as probes to detect specific DNA segments. PCR can be
used
to enhance the sensitivity of this method. PCR is an enzymatic amplification
directed
by sequence-specific primers, and involves repeated cycles of heat
denaturation of the

DNA, annealing of the complementary primers and extension of the annealed
primer
with a DNA polymerase. This results in an exponential increase of the target
DNA.
Other nucleotide sequence amplification techniques may be used, such as
ligation-mediated PCR, anchored PCR and enzymatic amplification as would be
understood by those skilled in the art.
(ii) Proteins

The C4H protein may be detected in a sample using antibodies that bind to the
protein as described in detail above. Accordingly, the present invention
provides a
method for detecting a C4H protein comprising contacting the sample with an
antibody that binds to C4H which is capable of being detected after it becomes
bound
to the C4H in the sample.

Antibodies specifically reactive with C4H, or derivatives thereof, such as
enzyme conjugates or labeled derivatives, may be used to detect C4H in various
samples, for example they may be used in any known immunoassays which rely on
the
binding interaction between an antigenic determinant of C4H, and the
antibodies.
Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g.
ELISA), immunofluorescence, immunoprecipitation, latex agglutination,
hemagglutination and histochemical tests. Thus, the antibodies may be used to
detect
and quantify C4H in a sample. In particular, the antibodies of the invention
may be
used in immuno-histochemical analyses, for example, at the cellular and sub-
subcellular level, to detect C4H, to localise it to particular cells and
tissues and to
specific subcellular locations, and to quantitate the level of expression.


CA 02510950 2005-06-14

28
Cytochemical techniques known in the art for localizing antigens using light
and electron microscopy may be used to detect C4H. Generally, an antibody of
the
invention may be labelled with a detectable substance and C4H may be localized
in
tissue based upon the presence of the detectable substance. Examples of
detectable
substances include various enzymes, fluorescent materials, luminescent
materials and
radioactive materials. Examples of suitable enzymes include horseradish
peroxidase,
biotin, alkaline phosphatase, (3-galactosidase, or acetylcholinesterase;
examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; and
examples
of suitable radioactive material include radioactive iodine 1-125, I-131 or 3-
H.
Antibodies may also be coupled to electron dense substances, such as ferritin
or
colloidal gold, which are readily visualised by electron microscopy.
Indirect methods may also be employed in which the primary antigen-antibody
reaction is amplified by the introduction of a second antibody, having
specificity for
the antibody reactive against C4H. By way of example, if the antibody having
specificity against C4H is a rabbit IgG antibody, the second antibody may be
goat
anti-rabbit gamma-globulin labelled with a detectable substance as described
herein.
Where a radioactive label is used as a detectable substance, C4H may be
localized by autoradiography. The results of autoradiography may be
quantitated by
determining the density of particles in the autoradiographs by various optical
methods,
or by counting the grains.
The ACD evaluation on the plant sample can be conducted using techniques
known in the art including the methodology described in Example 2.
(d) C4H Modulators
In addition to antibodies and antisense oligonucleotides described above,
other
substances that modulate C4H expression or activity may also be identified.
(i) Substances that Bind C4H
Substances that affect C4H activity can be identified based on their ability
to
bind to C4H.
Substances which can bind with the C4H of the invention may be identified by
reacting the C4H with a substance which potentially binds to C4H, and assaying
for


CA 02510950 2005-06-14

29
complexes, for free substance, or for non-complexed C4H, or for activation of
C4H.
In particular, a yeast two hybrid assay system may be used to identify
proteins which
interact with C4H (Fields, S. and Song, 0., 1989, Nature, 340:245-247).
Systems of
analysis which also may be used include ELISA.

Accordingly, the invention provides a method of identifying substances which
can bind with C4H, comprising the steps of:
(a) reacting C4H and a test substance, under conditions which allow for
formation of a complex between the C4H and the test substance, and
(b) assaying for complexes of C4H and the test substance, for free
substance or for non complexed C4H, wherein the presence of
complexes indicates that the test substance is capable of binding C4H.
The C4H protein used in the assay may have the amino acid sequence shown
in Table 4 (SEQ.ID.NO.:2) or may be a fragment, analog, derivative, homolog or
mimetic thereof as described herein.
Conditions which permit the formation of substance and C4H complexes may
be selected having regard to factors such as the nature and amounts of the
substance
and the protein.
The substance-protein complex, free substance or non-complexed proteins may
be isolated by conventional isolation techniques, for example, salting out,
chromatography, electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To
facilitate the assay of the components, antibody against C4H or the substance,
or
labelled C4H, or a labelled substance may be utilized. The antibodies,
proteins, or
substances may be labelled with a detectable substance as described above.
C4H, or the substance used in the method of the invention may be
insolubilized. For example, C4H or substance may be bound to a suitable
carrier.
Examples of suitable carriers are agarose, cellulose, dextran, Sephadex,
Sepharose,
carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic
film,
plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer,
amino
acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier
may be
in the shape of, for example, a tube, test plate, beads, disc, sphere etc.


CA 02510950 2005-06-14

The insolubilized protein or substance may be prepared by reacting the
material with a suitable insoluble carrier using known chemical or physical
methods,
for example, cyanogen bromide coupling.
The proteins or substance may also be expressed on the surface of a cell using
5 the methods described herein.
The invention also contemplates assaying for an antagonist or agonist of the
action of C4H.
It will be understood that the agonists and antagonists that can be assayed
using the methods of the invention may act on one or more of the binding sites
on the
10 protein or substance including agonist binding sites, competitive
antagonist binding
sites, non-competitive antagonist binding sites or allosteric sites.
The invention also makes it possible to screen for antagonists that inhibit
the
effects of an agonist of C4H. Thus, the invention may be used to assay for a
substance
that competes for the same binding site of C4H.

15 (ii) Peptide Mimetics
The present invention also includes peptide mimetics of the C4H protein of the
invention. Such peptides may include competitive inhibitors, enhancers,
peptide
mimetics, and the like. All of these peptides as well as molecules
substantially
homologous, complementary or otherwise functionally or structurally equivalent
to
20 these peptides may be used for purposes of the present invention.
"Peptide mimetics" are structures which serve as substitutes for peptides in
interactions between molecules (See Morgan et al (1989), Ann. Reports Med.
Chem.
24:243-252 for a review). Peptide mimetics include synthetic structures which
may or
may not contain amino acids and/or peptide bonds but retain the structural and
25 functional features of a peptide, or enhancer or inhibitor of the
invention. Peptide
mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl.
Acad,
Sci USA 89:9367); and peptide libraries containing peptides of a designed
length
representing all possible sequences of amino acids corresponding to a peptide
of the
invention.
30 Peptide mimetics may be designed based on information obtained by
systematic replacement of L-amino acids by D-amino acids, replacement of side
chains with groups having different electronic properties, and by systematic


CA 02510950 2005-06-14

31
replacement of peptide bonds with amide bond replacements. Local
conformational
constraints can also be introduced to determine conformational requirements
for
activity of a candidate peptide mimetic. The mimetics may include isosteric
amide
bonds, or D-amino acids to stabilize or promote reverse turn conformations and
to
help stabilize the molecule. Cyclic amino acid analogues may be used to
constrain
amino acid residues to particular conformational states. The mimetics can also
include
mimics of inhibitor peptide secondary structures. These structures can model
the 3-
dimensional orientation of amino acid residues into the known secondary
conformations of proteins. Peptoids may also be used which are oligomers of N-
substituted amino acids and can be used as motifs for the generation of
chemically
diverse libraries of novel molecules.
(e) Modulating C4H Expression
The present invention also includes methods of modulating the expression
and/or activity of the C4H gene or protein. Accordingly, the present invention
provides a method of modulating C4H expression or activity comprising
administering to a cell or plant in need thereof, an effective amount of agent
that
modulates C4H expression and/or activity. The present invention also provides
a use
of an agent that modulates C4H expression and/or activity.
The term "agent that modulates C4H expression and/or activity" or "C4H
modulator" means any substance that can alter the expression and/or activity
of the
C4H gene or protein. Examples of agents which may be used include: a nucleic
acid
molecule encoding C4H; the C4H protein as well as fragments, analogs,
derivatives or
homologs thereof; antibodies; antisense nucleic acids; peptide mimetics; and
substances isolated using the screening methods described herein.
The term "effective amount" as used herein means an amount effective, at
dosages and for periods of time necessary to achieve the desired results.
The term "plant" as used herein includes all members of the plant kingdom,
and is preferably an edible plant such as root vegetables or fruit. In a
preferred
embodiment, the plant is potato, apple or pear.
In one embodiment, the C4H modulator is an agent that enhances the
expression and/or activity of the C4H gene or protein. Enhancing the
expression of
the C4H gene can lead to enhanced production of chlorogenic acid which is one
of the


CA 02510950 2005-06-14

32
major phenolic compounds in plants that is involved in plant defense functions
against bacterial and viral pathogens. Enhancing C4H gene expression therefore
can
lead to enhanced disease resistance to these pathogens. Enhancing chlorogenic
acid is
also useful as it is a natural antioxidant and plants containing enhanced
levels will be
of greater nutritional value.
Accordingly, the present invention also provides a method of enhancing the
production of chlorogenic acid comprising administering an effective amount of
an
agent that enhances C4H gene expression or protein activity to a cell or plant
in need
thereof. Agents that enhance C4H gene expression or protein activity include
nucleic

acid molecules encoding the C4H protein, C4H protein as well as fragments,
analogs,
derivatives or homologs thereof. In a specific embodiment, the C4H nucleic
acid has
the sequence shown in Table 3 (SEQ ID. No.1) and the C4H protein has a
sequence
shown in Table 4 (SEQ ID. No. 2).
In another embodiment, the C4H modulator is an agent that decreases C4H
gene expression and/or C4H protein activity. Inhibiting C4H expression can be
used
to decrease ACD in plants as there is correlation between increased C4H levels
and
increased ACD in plants.
Accordingly, the present invention provides a method of decreasing ACD in
plants comprising administering and effective amount of an agent that can
inhibit the
expression of the C4H gene and/or inhibit the activity of the C4H protein.
Substances
that can inhibit the expression of the C4H gene include antisense
oligonucleotides.
Substances that inhibit the activity of the C4H protein include peptide
mimetics, C4H
antagonists as well as antibodies to C4H.

The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1
Materials and Methods
Potato Tuber Samples
Cultivars Russet Burbank and Russet Norkotah potato tubers were harvested
in 2002 from the Nova Scotia Agricultural College research field in Truro,
Nova


CA 02510950 2005-06-14

33
Scotia. The tubers were initially stored at 15 C for 14 d at 95% relative
humidity to
promote suberization. The temperature was then gradually decreased over a one-
month period to the final storage temperature of 9 C. Two diploid clones,
10908.06
and CH72.03, known for high and low degrees of ACD respectively, were obtained
from the Potato Research Centre, Agriculture and Agri-Food Canada in
Fredericton,
New Brunswick and stored at 9 C until needed.

Genomic DNA and Total RNA Isolation
Genomic DNA was isolated from Russet Burbank potato tubers using protocol
of Doyle and Doyle (1990) with minor modifications. Total RNA was isolated
from
potato tubers according to the slightly modified method of Bachem et al.
(1996). In
preparation for RNA isolation, selected potato tubers were peeled and the
cortex
region removed. The remaining tuber tissue was cut into 1 cm cubes and
immediately
frozen in liquid nitrogen. The frozen tissue was then ground to a fine powder
in
liquid nitrogen and stored at -80 C, until the total RNA was extracted.

Primer Design and Sequences
Primers for PCR were designed based on highly conserved regions between
the c4h cDNA of red pepper (Capsicum annuum) and c4h ESTs in tomato
(Lycopersicom esculentum) (Table 1) and synthesized by Invitrogen Canada (ON,
Canada).

Touchdown PCR
Touchdown PCR was used to amplify c4h gene from genomic DNA as the
primers were designed based on sequences of another species and there were
suspected mismatched nucleotides between the primer and the target sequences
(Sambrook and Russell, 2001b). The PCR reaction mixture contained the
following
components in a final volume of 25 yiL: 50 ng of genomic DNA, 2.5 pL of lOx
PCR
buffer, 1 ycL of 10 mM dNTPs, 1.5 U FastStart Taq (Roche Applied Science, PQ,
Canada), and 1 ysL of each primer (0.01 mM). PCR cycle parameters were as
follows: denaturation for 10 min at 95 C, followed by 30 cycles of denaturing
for 20
sec at 94 C, annealing at 60 C for 30 sec but after the first cycle the
annealing


CA 02510950 2006-09-14

= 34
temperature decreases by 0.5 C every cycle there after, and extension at 65 C
for I
min, following which there was another 30 cycles of 94 C for 20 sec, 45 C for
30 sec,
and 65 C for 1 min. There was then a final extension for 7 min at 72 C. The
extension time during the cycle was changed from I min to 2 min depending on
the
length of the amplicon expected. If the amplified region was expected to be
more
than 2 kb in length, an extension time of 2 min was used.

Reverse Transcriptase-PCR (RT-PCR)
The two-step reverse transcriptase-PCR (RT-PCR) method was used to
amplify the c4h coding region from synthesized cDNA, as described in the
Eppendorf
Waster RTp1usPCR kit (VWR International, PQ, Canada). The first step of the
two-
step method was the synthesis of first strand cDNA from total RNA. The second
step
of the Eppendorf Waster RTplusPCR kit was the PCR amplification of the first
strand cDNA. Primers at the 5' and 3' ends for RT-PCR reactions were designed
based on the sequenced genomic DNA (5' primer: 5'-atggatcttctcttactggag-3'
(SEQ
ID NO: 3); 3' primer: 5'-ggtttacacaaacaaacaac-3'(SEQ ID NO: 4)).

5'and3'RACE
The protocol followed is as described in the 5'/3' RACE kit (Roche Applied
Science, PQ, Canada). The primers used in this experiment are shown in Table
2,
along with their sequences and melting temperatures. The primers labeled as SP
are
sequence specific primers designed against sequenced regions of the c4h gene
in
potato. The first strand cDNA was purified using the High Pure PCR Product
Purification kit (Roche Applied Science, PQ, Canada) to remove unincorporated
nucleotides and primers as per manufacturer's instructions. The addition of a
homopolymeric A-tail to the 5' end of the cDNA was carried out by the enzyme
terminal transferase (provided by the kit).
For rare mRNA such as for c4h, a second round of PCR with nested primers
was required to obtain a visible PCR product. The nested sequence specific
primer
(SP3) was designed 75 bp within the previously amplified region, while the
reverse
nested primer was complementary to the Oligo dT-anchor primer (provided with
the
5'/3' RACE kit). As the concentration of the amplified dA-tailed cDNA product
was


CA 02510950 2006-09-14

unknown, the nested PCR was performed on both undiluted and diluted (1:20 in
water) amplified product. The PCR conditions for amplification were identical
to the
conditions used in the first round of amplification.
The method of 3' RACE (Rapid Amplification of cDNA Ends) takes
5 advantage of the naturally occurring poly(A) tail of mRNA to amplify the 3'
end of a
gene. The 5'13' RACE kit (Roche Applied Science, PQ, Canada) was used to
synthesize first strand cDNA and amplify the 3' end using both sequence
specific
primers and supplied primers.
Cloning of PCR Products into a Plasmid Vector
10 Media for transformation were made according to standard protocols
(Sambrook and Russell, 2001c). PCR reaction mixtures were filtered through the
Millipore Ultrafree-DA filter kit (Fisher Scientific, ON, Canada) to remove
salts,
unincorporated dNTPs, and primers. The ligation of the PCR product to the pGEM
-
T vector was set up according to manufacturer's instructions (Promega Corp.,
WI,
15 USA). The protocol for the production of E. coli DH5_ competent cells and
the
subsequent transformation was followed as described by Inoue et al. (1990).

DNA Sequencing and Data Alignment
All sequencing of plasmid DNA and PCR products was performed by DNA
20 Landmarks, Inc. (PQ, Canada). The universal primers, T7 (5'-
TAATACGACTCACTATAGGG-3' (SEQ ID NO: 5)) and SP6 (5'-
GATTTAGGTGACACTATAG-3' (SEQ ID NO: 6)) were used for sequencing of the
pGEM -T plasmid DNA constructs. Raw sequence chromatograms were visually
edited using the software program Chromas (<http://www.technelysitim.com.au>).
25 Alignment of the sequence data was conducted using the BLAST program
available
from the National Center for Biotechnology Information
(<http://www.ncbi.nlm.nih.gov>) (Altschul et al., 1990).

Northern Hybridization
30 The probe used for Northern hybridization was a 550 bp cDNA fragment
previously amplified using the primers AF and FR (see Table 1) from the Russet
Burbank total RNA. The fragment was subcloned into the pGEM -T vector, as
described above. The QlAfilter Plasmid Maxi kit (Qiagen, ON, Canada) was used
to


CA 02510950 2005-06-14

36
isolate plasmid DNA on a large scale from transformed cells as per
manufacturer's
instructions. The restriction enzyme Pvu II was used to release the insert
from the
vector as it cuts outside of the T7 and SP6 promoter/primer sites of the
vector, which
are necessary for PCR amplification and transcription.
The c4h probe was then PCR amplified using the T7 and SP6 primers using
standard PCR conditions. The amplified product was filtered through the
Millipore
Ultrafree-DA filter kit, prior to labeling. The probe was radioactively
labeled using
the DECAprimeTM II Random Priming DNA Labeling kit (Ambion, TX, USA). For
use as a positive control, the c4h probe was transcribed to RNA (Sambrook and
Russell, 2001a) and loaded to the denaturing gel along with the samples. Any
DNA
remaining was degraded by incubating for 15 min at 37 C with 20 U of DNase I,
after
which it was diluted to 1:100 and 1:1000 and stored at -20 C in 10 L aliquots.
RNA from Russet Burbank and Russet Norkotah potato tubers as well the
RNA from identified ACD-dark (10908.06) and ACD-light (CH72.03) diploid clones
were analyzed. To ensure that the pattern and intensity of the hybridization
signal
measured for each of the four tuber samples was reproducible, the Northern
hybridization experiment was replicated three times using identical
conditions.
Denaturing gel electrophoresis and membrane transfer of the RNA samples
(30 g total RNA/sample) was performed according to standard protocols
(Sambrook
and Russell, 2001a). Prehybridization of the membrane was performed in 6 mL of
Ultrahyb hybridization buffer (Ambion, Inc., TX, USA) for 1 h at 42 C. The
buffer
was replaced with 6 mL of new Ultrahyb solution containing the denatured
probe.
Hybridization of the membrane was performed overnight at 42 Cb. Following
hybridization, the membrane was gently rinsed in 5x SSC at room temperature,
then
washed 2x for 5 min each at 37 C in 5x SSC with 0.1% SDS, and lastly in 1x SSC
with 0.1% SDS 2x for 15 min each at 37 C. The membrane was exposed to Kodak
BioMax XAR film (Fisher Scientific, ON, Canada) 5 to 10 d prior to developing.
The
film was developed using Kodak GBX Developer and Fixer (Fisher Scientific, ON,
Canada) as per manufacturer's instructions.
Evaluation of ACD in Cooked Potato Tubers
To correlate c4h transcript levels with the degree of tuber darkening; the ACD


CA 02510950 2005-06-14

37
levels of each potato cultivar used in the Northern hybridization experiment
were
digitally measured. As the length of tuber storage affects the degree of
darkening,
ACD levels of the tuber samples were measured at the same time period as the
tuber
samples were frozen for subsequent hybridization experiments. The level of

darkening was digitally measured based on a gray scale of 256 pixel units,
where
white has a pixel density of 255 and black a pixel density of 0. Therefore, a
higher
pixel density was indicative of a lighter tuber while a lower pixel density
corresponded to a darker tuber.
Four tubers for each tetraploid cultivar and two tubers for each diploid clone
were cooked by steaming, sliced lengthwise, and the cut surfaces exposed to
air for 1
h to allow darkening to develop. For the tetraploid cultivars, the pixel
density of the
entire surface of four tuber halves (one half from each tuber) was measured.
As there
were a limited number of tubers available for the diploid clones, the pixel
density of
both halves of each of the two cooked tubers was measured. The images of four
tuber
surfaces for each sample were captured and digitally evaluated using the UVP
Chemi-
Imager System and LabWorks Imaging Analysis and Acquisition Software.
The four ACD measurements for each tuber cultivar/clone were analyzed by a
one-way analysis of variance using the Proc GLM (General Linear Model)
procedure
of SAS (Version 8, SAS Institute, NC, USA). Significance at the 5% level (P-
value <
0.05) was further examined using Tukey's honestly significant difference (hsd)
test (_
= 0.05) to compare the mean pixel densities. Normality and constant variance
were
tested using the Proc Univariate procedure of SAS using the predicted and
residual
values. The data proved to be normal without transformation of the data.

Results
The c4h Gene in Potato
The analysis of the sequencing data generated from the genomic DNA and
cDNA clones led to the identification of the 5'- and 3'-UTR, three exons, and
two
introns from the 2986 bp DNA sequence of the potato genome. The complete c4h
gene sequence is shown in Table 3. The coding region of the gene is 1518 bp in


CA 02510950 2005-06-14

38
length, starting at the ATG nucleotides at positions 45 to 47 and stopping at
nucleotides TAA at positions 2861 to 2863. The coding region contains three
exons
(shown in bold) and two introns. Exon 1 is 785 bp in length (from nucleotide
45 to
829), exon 2 is 134 bp (from nucleotide 1363 to 1496), while exon 3 is 599 bp
(from
nucleotide 2265 to 2863). Intron 1 and 2 are 533 and 768 bp, respectively.
Intron 1 is
located between nucleotides 785 and 786 of the coding region and intron 2 is
located
between nucleotides 919 and 920. The partially sequenced 5'- and 3'-UTR
measures
44 (positions 1 to 44) and 123 bp (position 2864 to 2986) in length,
respectively.
The C4H protein sequence is composed of 505 amino acids (not including the
stop codon), as shown in Table 4. The typical start (ATG) and stop (TAA)
codons are
found at the beginning and end of the open reading frame. The splice sites for
introns
1 and 2 are depicted by arrows in Table 4. Intron 1 is found between the
second and
third nucleotides of the codon (AA/G), which corresponds to the amino acid
lysine
(amino acid position 262). Intron 2 is positioned between the first and second
nucleotides of the codon (G/CA), which codes for the amino acid alanine (amino
acid
position 307).

The Gene Expression of c4h in Tuber Tissues
The c4h hybridization signals in the four tuber samples are shown in Figure
IA. The size of the c4h transcript was measured based on the location and
known size
of the transcribed c4h probe (710 bp) and the 25S, 18S, and 5S ribosomal RNA
bands
(3.8 kb, 2.0 kb, 0.74 kb, respectively). The size of the c4h signal is between
600 and
800 bp (Figure 1B). The intensity of the c4h transcript for each potato sample
was
measured relative to the Russet Burbank signal, which was assigned a value of
1.0,
using Labworks Imaging and Acquisition Software.
In order to verify that there was equal loading of the total RNA used for
Northern hybridization, the ribosomal RNA (both the 18S and 25S bands) was
quantified digitally from an underexposed agarose gel image (not shown), like
that
shown in Figure 1B, prior to transferring the RNA to the membrane. The Russet
Burbank 18S and 25S ribosomal RNA bands were assigned a value of 1Ø The
relative intensities of the remaining three samples were given a value
relative to the
Russet Burbank RNA. The relative intensities of the c4h transcript were
adjusted


CA 02510950 2005-06-14

39
according to the proportion of RNA loaded for each sample. The mean adjusted
c4h
transcript levels of the four tuber samples are shown in Table 5. The mean c4h
transcript levels are significantly different between the dark and light
diploid samples
with 1.70 and 0.91, respectively. The intensity of the c4h hybridization
signal was
not significantly different between the dark diploid sample and the two
tetraploid
cultivars. As well, there were no significant differences among the tetraploid
cultivars and the light diploid sample.
The potential relationship between ACD and c4h gene expression was
analyzed by comparing the mean relative c4h transcript levels to the mean
degree of
darkening previously measured for each tuber sample (Figure 2). The mean pixel
densities of Russet Burbank and Russet Norkotah tubers were found to be 113
and
114, respectively, which was not significantly different at _ = 0.05. For the
light
diploid clone CH72.03, the mean pixel density was slightly higher (121 pixel
units),
which corresponded to a lower degree of ACD. The mean pixel density of the
dark
diploid clone 10908.06 was found to be significantly lower at 89 pixel units,
which
corresponds to a higher level of tuber darkening.
As seen in Figure 2, there are no significant differences in the degree of ACD
or in intensity of the c4h transcript in Russet Burbank and Russet Norkotah.
In
contrast, the degree of darkening in the identified dark diploid sample is
significantly
higher when compared to the identified light diploid, and the relative
intensity of the
c4h hybridization signal is also significantly higher in the dark diploid when
compared to the light diploid. These results suggest that the level of the c4h
transcript
may determine the susceptibility of the tuber to ACD.

Discussion

In this study, the 2986 bp c4h gene was sequenced from the potato cultivar
Russet Burbank. This full-length sequence included the coding region, two
introns,
and partial 5'- and 3'-UTR. The c4h gene has been sequenced in a number of
plant
species, however it had not previously been cloned from the potato genome. The
coding sequence for the potato c4h gene, from the start codon (ATG) to the
stop
codon (TAA), is 1518 bp in length.

The similarity of class I c4h coding sequences from other plant species to the


CA 02510950 2005-06-14

potato c4h coding sequence is shown in Table 6. The nucleotide sequence of red
pepper is the most similar to potato at 91% (1379 bp of 1518 bp), which was
anticipated as both belong to the Solanaceae family. The sequence similarity
of the
remaining plant species to the potato c4h coding sequence, range from 82 to
67%.

5 The c4h coding region in potato is 1518 bp in length, which is identical to
the
length of most other class I c4h coding regions (Table 6). From the species
listed in
Table 6, only three were of a different length when compared to potato. The
coding
region of alfalfa and Bishop's weed are 3 bp longer (1521 bp), corresponding
to an
additional amino acid. In alfalfa, the three extra nucleotides (codon GAT)
occur at
10 positions 848 to 850 of the coding sequence; translating to the amino acid
aspartate at
position 274 of the peptide sequence. Conversely, the additional amino acid in
the
Bishop's weed peptide sequence is a methionine occurring at position 1, after
translation. This corresponds to the nucleotide codon ATG, which is followed
by the
typical ATG start codon. Sweet orange is the third plant species showing a

15 significantly longer c4h coding region, with a length of 1560 bp. The sweet
orange
c4h coding region contains an additional 42 bp, corresponding to fourteen
amino
acids. The sweet orange c4h gene carries a unique N-terminus from nucleotides
10 to
106, which not only contains the 42 additional nucleotides but also shows no
homology to this region in other c4h genes.
20 In potato, the three individual exons are 785, 134, and 599 bp which
together
make up the complete c4h coding region. The lengths of the c4h gene have only
been
cloned from genomic DNA in Arabidopsis, pea (Pisum sativum), Japanese aspen,
and
French bean (Phaseolus vulgaris) (Kawai et al., 1996; Bell-Lelong et al.,
1997;
Nedelkina et al., 1999; Whitbred and Schuler, 2000). The first three are class
I c4h
25 genes, whereas the c4h gene from French bean is class II. Figure 3 depicts
the
positions and lengths of the exons and introns in the four plant species as
well as in
potato. The similarity between the exon lengths of potato, Arabidopsis, pea,
and
Japanese aspen would suggest that there is a conserved splice position in all
class I
c4h genes. When compared, it was found that the nucleotides flanking the
splice site
30 positions for both introns were identical in all four plant species (Figure
3). The
splice site for intron 1 occurred between the second and third nucleotides of
the codon
AA/G at positions 785 and 786 of the coding region. The codon, disrupted by
intron


CA 02510950 2005-06-14

41
1, corresponds to the amino acid lysine at position 262 of the protein
sequence, Table
4. The second intron excision site is between nucleotides 919 and 920 of the
coding
region at codon G/CA. This codon represents the amino acid alanine at a
position of
307 (Table 4). The class II c4h gene from French bean, differs from the others
as it
only contains two exons and one intronic region.
Two introns corresponding to those in the c4h gene were cloned from the
potato genome. The lengths of the introns are 533 and 768 bp for the first and
the
second introns, respectively. Although the c4h gene in potato, Arabidopsis,
pea, and
Japanese aspen each contains the same number of introns, seemingly identical
intron

splice sites, and homologous coding regions; there is no homology among the
intron
sequences. Furthermore, a comparison of the potato c4h intron sequences to the
entire Genbank database resulted in no sequence matches. The lack of homology
for
the c4h introns is also reflected in the differences between the lengths of
the introns.
The intron sizes appear to relate to the complexity and size of the
corresponding plant genome. Evidence to this effect is demonstrated by the pea
c4h
gene, which has one of the largest introns (1726 bp). The genome size of pea
is 4.8 x
109 bp, making it one of the largest and most complex genomes in the plant
kingdom
(Ellis and Poyser, 2002). In contrast, Arabidopsis has the shortest introns
and the
smallest plant genome at 1.1 x 108 bp (The Arabidopsis Genome Initiative,
2000).
Japanese aspen and potato fall into the mid-range with introns of
approximately 500
to 800 bp and genome sizes of between 5.0 x 108 bp and 8.7 x 108 bp,
respectively
(Arumuganathan and Earle, 1991; Taylor, 2002). Previous studies have shown
weak
correlations between intron and genome size in eukaryotes, including humans,
Drosophila, and Japanese pufferfish (Fugu rubripes) (Moriyama et al., 1998;
McLysaght et al., 2000). There is little information on the correlation of
intron size to
genome size in plant species but it has been suggested that plants with small
genomes
also have smaller introns (Deutsch and Long, 1999). Conversely, it was
demonstrated
that different sized genomes in cotton species had no impact on intron size
(Wendel et
al., 2002).
The 5'-untranslated region (5'-UTR) is the region from the transcriptional
initiation site to the start codon for translation. Previously, it was
reported that the
average length of the 5'-UTR in plants was 168 bp, while the average length in
dicot


CA 02510950 2005-06-14

42
species was 98 bp (Pesole et al., 1997; Kochetov et al., 2002). The complete
5'-UTR
for c4h is only available in two other plant species: Arabidopsis and French
bean
where it was measured to be 86 and 78 bp, respectively. In comparison the 5'-
UTR
for the potato c4h gene was only 44 bp in length. This indicates that either
the potato
c4h 5'-UTR is much shorter than the average or that only the partial 5'-UTR in
potato
has been sequenced. Alignment of the 5'-UTR sequences from Arabidopsis, French
bean, and potato has found that there is no similarity between any of the
three
sequences. A search of the Genbank database for sequence similarity to the
potato
5'-UTR also did not result in any significant similarities to any other 5'-
UTRs. This
makes it difficult to determine whether the one sequenced in potato is the
entire 5'-
UTR.

A sequence characteristic of the 5'-UTR is the pronounced imbalance between
the levels of GC and AU. The partial c4h 5'-UTR in potato also demonstrates
this
imbalance with a GC content of 34.1% and an AU content of 65.9%. This is
comparable to the average GC content in the 5'-UTR of dicot species of 39%
(Kochetov et al., 2002). This low GC content reduces secondary structures
allowing
translational efficiency to be improved (Groenewald et al., 2000; Kochetov et
al.,
2002).

In this study, a 123 bp 3'-UTR was sequenced for the potato c4h gene. In
plants, this region is thought to be much more variable in length among
species than
the 5'-UTR, ranging from 240 to 740 bp (Pesole et al., 1997). The GC content
of the
c4h 3'-UTR in potato is the lowest at 24.4%, when compared to other regions of
the
gene. A review on plant 3'-UTRs showed that the GC content is the lowest in
the 3'-
UTR (35%) when compared with other regions of the plant genome (Pesole et al.,
1997). The c4h 3'-UTR GC content is much lower than the reported value however
it
would seem that every segment of the c4h gene contains a lower GC content when
compared to published literature. A BLAST comparison of the 3'-UTR to other
nucleotide sequences in Genbank , resulted in only the c4h 3'-UTR from red
pepper
(Accession If AF212318) showing any homology. The sequence alignment of the
123
bp 3'-UTR from potato resulted in a match of 92 bp out of the first 123 bp in
the red
pepper 3'-UTR. In red pepper the 3'-UTR has a length of 219 bp, ending with
the
conserved polyadenylation signal. It is then believed that the 3'-UTR in
potato is


CA 02510950 2006-09-14

43
only partially cloned, since the polyadenylation signal was not identified at
the 3' end.
The C4H protein consists of 505 amino acids, excluding the stop codon (Table
4). The alignment of C4H amino acid sequences showed high homology between
potato and the same protein in many other plant species, as seen in Table 7.
The
highest sequence similarity to potato was shown to be the red pepper C4H amino
acid
sequence at 87%. The other 14 plant species were very similar to the potato
C4H
amino acid sequence with a similarity of between 84 and 80%.
A comparison of the potato C4H amino acid sequence to C4H sequences from
other plant species allows homologous regions and domains unique to the CYP73
protein family to be identified. The alignment of six C4H sequences showing
high
similarity to potato (red pepper, lithospermum, Madagascar periwinkle, tree
cotton,
wild licorice, and poplar) is shown in Table 8 (SEQ ID NOS: 2, 29-34,
respectively,
in order of appearance). The first domain is a hydrophobic region at the N-
terminus
from position 3 to 23, represented by Box A in Table 8 (Ro et al., 2001). This
region
is responsible for membrane binding, protein stability, and is a signal-anchor
to keep
the protein on the cytoplasmic side of the endoplasmic reticulum (Hotze et
al., 1995;
Nedelkina et al., 1999). Among the plant species shown in Table 8, the
majority of
the substitutions involve the same five amino acids, isoleucine (I), valine
(V), leucine
(L), phenylalanine (F), and alanine (A), which are all hydrophobic in nature.
This
indicates that the presence of hydrophobic amino acids is partly responsible
for the
function of this domain, rather than the presence of specific amino acids.

The second domain is a proline rich region which occurs from amino acid 34
to 41 of the C4H protein sequence (Box B in Table 8). This region is thought
to be
responsible for correctly orientating and folding the protein in the membrane
by
breaking a-helix bonds (Mizutani et al., 1997; Koopmann et al., 1999).

Cytochrome P450 proteins contain a conserved region that is involved in the
binding and activation of dioxygen, which is necessary for oxygen
incorporation into
the corresponding substrates (Schalk et al., 1999). The consensus sequence for
this
region in plant P450 proteins is as follows; (A/G)(A/G)I(E/D)T. As seen in
Table 8
(Box C), the sequence of the motif in the C4H protein family is AAIET and is
identical in all plant species shown.

One of the most important domains in the P450 family of proteins is the heme-


CA 02510950 2005-06-14

44
binding domain positioned at amino acids 439 to 449 (Box D of Table 8). The
consensus sequence for this domain in P450 proteins is PFGXGRRXCXG. In the
CYP73 family, the domain (PFGVGRRSCPG) is conserved in all plant species
sequenced, indicating that there is a consensus sequence specifically for C4H.
The
importance of this domain is that it allows the binding of the heme molecule
to the
enzyme which is essential for catalysis and the ability to bind carbon
monoxide
(Chapple, 1998). The binding of the heme molecule occurs through a thiolate
side
chain that originates from the conserved cysteine amino acid at position 447
(Schalk
et al., 1999). In the C4H family of proteins, the interaction of the conserved
cysteine
(C) and the subsequent proline (P) molecule enables the formation of a
"cysteine
pocket" in which the sulfur-iron bond is in the center of a hydrophobic
environment
(Schalk et al., 1999).
The objective of the Northern hybridization analysis was to determine if
changes in c4h gene expression occurred in cultivars with varying degrees of
ACD.
The results of the Northern hybridization and the ACD evaluation data suggest
that

potentially there is a relationship between c4h gene expression and ACD, as
seen in
Figure 1. The level of the darkening for Russet Burbank and Russet Norkotah
tubers
was similar at 113 and 114 mean pixel density, respectively. Russet Norkotah
generally is considered darker than Russet Burbank based on other researcher's
observations (Wang-Pruski, personal communication), but samples from this
growing
location and year provided very similar ACD levels for both samples. As shown
in
Figure 1, the mean relative intensity of c4h transcript in Russet Burbank and
Russet
Norkotah was also not significantly different.
The mean pixel density of the two tetraploid cultivars was significantly lower
when compared to the light diploid clone. However, the mean relative intensity
of the
c4h transcript in the tetraploid cultivars was not significantly different to
the light
diploid clone. Although the dark diploid clone had a significantly lower mean
pixel
density as compared to the tetraploid cultivars; it was not significantly
different in
terms of the c4h transcript intensity. The mean relative intensity of the dark
diploid
sample was much higher than the other samples tested, however because of the
high
variability among the three replicates for the dark diploid clone (as shown by
the
standard error bars on Figure 2) there was no significant differences to the
tetraploid


CA 02510950 2005-06-14

cultivars. Finally a comparison of the two diploid clones showed that the
identified
light diploid clone had a significantly higher mean pixel density (lower ACD
susceptibility) and had significantly lower c4h transcript intensity. On the
other hand,
the identified dark diploid clone had a significantly lower mean pixel density
(higher
5 ACD susceptibility) and demonstrated significantly higher c4h transcript
intensity.
The lack of significant differences between the tetraploid cultivars for ACD
and c4h transcript intensity, as well as the significant differences in
intensity for the
dark and light diploid clones suggests that c4h is involved in the ACD
mechanism.
This evidence indicates that there is a possible relationship between c4h gene
10 expression levels and the level of darkening in the tuber, where potato
cultivars with
higher c4h expression levels have an increased susceptibility to ACD, and vise
versa.
The Northern hybridization results show that the size of the signal measured
does not match the full-length 1.5 kb c4h transcript. The transcript detected
is 600 to
800 bp in length, which is much shorter than the full-length cDNA cloned in
this
15 study. It is unlikely that non-specific hybridization occurred, as the
probe was
homologous to only those sequences encoding c4h in Genbank and the transcript
was detected at the same position in each of the four samples. Degradation of
the
RNA is not likely since electrophoresis of the total RNA used in the Northern
hybridization experiment (Figure 1A) showed intact 25S and 18S bands. The
20 presence of intact and distinct 28S (25S in potato) and 18S ribosomal RNA
bands is
considered the simplest and best indicator of high quality RNA (Miller et al.,
2004;
Palmer and Prediger, 2004).

In this study, the hybridization probe was homologous to the 5' end of the
gene (from 100 bp to 572 bp of the coding sequence), which means any
degradation
25 occurring at the 3' end would not have been detected. The truncation of an
mRNA
transcript can be the result of controlled degradation (decay) of the mRNA,
alternative pre-mRNA splicing of exons/introns, or cleavage by microRNA
(miRNA).
All three of the above mechanisms are key in the regulation of gene expression
at the
mRNA level (Konig et al., 1998; Yu and Kumar, 2003).
30 Controlled degradation of the mRNA is the first possible mechanism for
creating truncated mRNA in order to regulate the gene expression levels. In E.
coli,
mRNA levels are regulated by 3' to 5' exonucleases or endonucleolytic
cleavage,


CA 02510950 2005-06-14

46
followed by 3' to 5' exonucleolytic degradation of the products (Belasco and
Higgins,
1988). Eukaryotic mRNA is polyadenylated and is degraded by first
deadenylation
and then degradation of the mRNA in a 3' to 5' direction. The speed of this
degradation determines the half-life of the mRNA molecule. The degradation of
eukaryotic mRNA is a fast and flexible form of posttranscriptional regulation
and
allows plants to adapt rapidly to changing conditions (Sullivan and Green,
1996).
In soybean and petunia, it was found that the degradation of mRNA encoding
ribulose-1,5-bisphosphate carboxylase (rbcS) occurred by endonuclease cutting
of the
full-length transcript at several specific sites in a 3' to 5' direction
(Tanzer and
Meagher, 1994). The degradation of SAUR (small auxin up RNA) transcripts in
soybean occurred within 10 to 50 min (Sullivan and Green, 1996). In
mitochondrial
transcripts analyzed from pea, it was found that the length of the poly(A)
tail
influences the rate of mRNA decay (Kuhn et at., 2001). A poly(A) tail composed
of
more than 10 adenine molecules results in the degradation of the full-length
transcript
after 10 min into multiple smaller products. When only 3 adenine molecules
comprised the poly(A) tail, no degradation of the transcript occurred after 60
min
(Kuhn et al., 2001).
Alternative pre-mRNA splicing of exons/introns is the second possible
mechanism for the truncation of mRNA transcripts. The regulation of
alternative
splicing is dependent on factors such as: developmental stage, tissue type,
and
response to various stimuli including growth factors, hormones, cytokines,
membrane
depolarization, and wounding (Konig et at., 1998). The alternative splicing of
a
transcript can often lead to premature termination of translation, altered
protein
structure, and a loss of protein stability or function. Alternative pre-mRNA
splicing
of the introns has resulted in the presence of truncated mature transcripts in
morning
glory (Ipomoea purpurea), peach (Prunus persica), and tobacco (Nicotiana
tabacum)
(Dinesh-Kumar and Baker, 2000; Bassett et at., 2002; Zufall and Rausher,
2003). In
morning glory, a large DNA insertion in an intronic region of the gene
encoding
flavonoid 3'-hydroxylase resulted in the mis-splicing of the pre-mRNA and a
corresponding shift in the open reading frame. The resulting transcript was
only 500
bp compared to the full-length transcript of 910 bp (Zufall and Rausher,
2003).
Alternative splicing of an intron in an ethylene receptor gene in peach
resulted in two


CA 02510950 2005-06-14

47
different length mature transcripts (Bassett et at., 2002). The longer of the
two
transcripts produced was found to be the most abundant in developing fruit,
suggesting that developmental processes regulate the alternative splicing of
this gene.
It seems unlikely that alternative splicing of the c4h transcript occurred, as
it would
probably lead to a loss of enzyme function. As the C4H enzyme is involved in
the
regulation of the phenylpropanoid pathway, it would be necessary for its
activity to be
maintained.
The final mechanism for truncation of the transcript is cleavage by miRNA.
Recent findings have suggested that 21 nt miRNA are involved in gene
expression
regulation in plants through miRNA-directed cleavage (Xie et at., 2003). Each
miRNA has an exact complementarity to the target mRNA. The miRNA binds to the
target where it directs the cleavage of the mRNA transcript at the binding
site (Floyd
and Bowman, 2004). To date, most of the targets identified are transcriptional
factors
that are crucial to cell growth and development (Ke et at., 2003). Rhoades et
at.

(2002) found that out of 49 predicted targets for miRNA-directed cleavage, at
least 34
encoded for known or putative transcription factors. Studies of miRNA-directed
cleavage have been reported in the model plant species, Arabidopsis. The
truncation
of mature transcripts by miRNA-directed cleavage is a possible mechanism for
posttranscriptional regulation. However, based on the limited studies
available it

seems as though miRNA-directed cleavage is a posttranscriptional form of
regulation
for transcriptional factors rather than functional genes, such as c4h.
Based on the evidence presented, the most likely mechanism for truncation of
the potato c4h transcript would be controlled degradation. Although it has not
been
reported previously in potato tubers, controlled mRNA degradation of c4h is
possible.
The degradation of the c4h transcript would lead to the detection of different
length
hybridization signals. Since the C4H enzyme is in such low quantities in plant
tissue;
its rapid rate of degradation would lead to difficulties in detecting the full-
length
transcript. Also, as C4H is a regulatory enzyme in the phenylpropanoid
pathway;
controlled mRNA degradation would be a fast and efficient way to regulate c4h
transcript levels during the biosynthesis of CA.


CA 02510950 2005-06-14

48
Example 2

The goal of this example was to study the differential gene expression of C4H
gene in ACD susceptible (dark) and ACD resistant (light) diploid potato clones
and
tetraploid cultivars that are involved in the ACD trait.

Differential gene expression analysis of C4H gene in ACD dark and light
clones of diploid families and tetraploid cultivars were performed using
relative
quantitative RT-PCR. Chlorogenic acid, citric acid, and chlorogenic acid to
citric acid
ratio in the selected samples were analyzed. Statistical methods were used to
find the
significant differences in differential gene expression data and chemical
concentration
data among ACD dark and ACD light samples.
Statistical analyses were performed to study the effect of ACD (dark and
light)
on the expressions of the C4H gene and the chemical concentration in the
samples.

MATERIALS AND METHODS
Potato samples

Potato clones of two diploid families and two tetraploid cultivars were used
in
this study. The clones of the diploid families used were the progenies of two
individual crosses between the ACD dark and ACD light parents. The clones
originated from one cross were named family 13610 and the clones originated
from
another cross were named family13395. The clones of the family 13610 grown at
the
research field at Nova Scotia Agricultural College, Truro, Nova Scotia was
named as
13610-T. The clones of the family 13395 were grown at Potato Research Centre,
Agriculture and Agri-Food Canada, Benton Ridge, Fredericton, New Brunswick was
named as 13395-B. The two tetraploid cultivars, Russet Burbank and Shepody
were
grown at the research field at Nova Scotia Agricultural College, Truro, Nova
Scotia.
All the tubers were grown and harvested in 2002 and 2003 season. Standard
field
practices were carried out for all the tubers. Only tubers from 2003 seasons
were
used for differential gene expression analyses.
The harvested tubers were packed in paper bags and subsequently stored in the
cold storage room, at 15 C and 95% relative humidity for two weeks at. The
storage
temperature was decreased gradually to 10 C over a month, tubers were finally
stored


CA 02510950 2005-06-14

49
at 9 C with 95% relative humidity.

ACD Evaluation

ACD evaluation was performed for all the tubers of the diploid families and
tetraploid cultivars. The ACD levels of the stored tubers were measured in
January.
That is, ACD levels for tubers harvested in year 2002 was carried out in 2003
and for
the tubers harvested in 2003 were performed in 2004. The January ACD
measurements of 2003 and 2004 were used for sample selection in this study.
ACD
evaluation was done using digital imaging (Wang-Pruski and Tarn, 2003) by a
lab
technician for both the years. Digital images of the cooked tuber surface were
taken
using a cooled CCD camera attached to the UVP Biochemi Imaging System (UVP
Inc., Upland, CA, USA). The LabWorksTM image acquisition and analysis software
(UVP Inc., Upland, CA, USA) was used for acquiring the digital image of the
cooked
tubers. The degrees of ACD in the cooked tubers were measured using mean raw
pixel density (MRD) at 0-255 pixel levels (where 0 is black and 255 is white).
Only
the potato clones with white flesh color were used.

Sample selection

The ACD levels were evaluated in all the tubers of diploid families for
January 2003 and January 2004. The tubers with the lowest MRD are considered
to
be susceptible to ACD (ACD dark) and the tubers with the highest MRD are
considered to be resistant to ACD (ACD light). The ACD values measured were
plotted in ascending order (lowest MRD to the highest MRD) against the
respective
clones of each diploid family under study. The ACD data collected from the two
years (January 2003 and January 2004) were correlated. Two or three diploid
clones
showing similar ACD values in both January 2003 and January 2004 were chosen.
Similarly the ACD measurements of tetraploid cultivars, Shepody and Russet
Burbank were measured in January 2003 and 2004.

Sample preparation

The selected potato tubers were carefully peeled to remove the outer skin. The


CA 02510950 2005-06-14

tubers were then cut in half and the center region including the pith was
carved out.
The outer layer (Figure 4), which was about 1cm in breadth, was chopped using
a
knife. The chopped tuber pieces were used for: 1) fresh tuber samples were
used for
the measurement of chlorogenic acid and citric acid by high performance liquid
5 chromatography (HPLC), and 2) the remaining tuber pieces were frozen in
liquid
nitrogen and stored at -80 C for total RNA extraction.

Primer design

The nucleotide sequences of the cDNA of selected candidate genes were
either obtained from Genbank (http://www.ncbi.nlm.nih.gov/), or from the
potato
10 molecular biology lab at NSAC. The obtained sequences were used to design
primers
using online primer designing software Primer3 (http://frodo.wi.mit.edu/cgi-
bin/primer3/primer3_www.cgi). The primers were selected based on the following
characteristics: the minimum primer length was 15 nucleotides, the product
size was
less than 700bp, melting temperature was between 55 C to 65 C, GC
concentration
15 was between 45% to 60%, base pair self complimentarity was less than 4
nucleotide
pairs, 3' complimentarity was less than 3 nucleotide pairs.

Total RNA extraction from potato tubers

Total RNA was extracted using the protocol followed by Singh et al. (2003).
The frozen potato tubers were ground with a presterilized pestle in a mortar
using
20 liquid nitrogen. About 300mg of the frozen tuber was transferred to a 2ml
microcentrifuge tube. To the frozen potato powder 500p,1 of extraction buffer
(50mM
Tris-HCI, pH 9.0; 150mM NaCl; 1% sarkosyl; 20mM EDTA; 5mM DTT) was added.
After vortexing the tube, 500 1 of phenol: chloroform:isoamyl alcohol
(25:24:1) was
added. The tube was vortexed again and centrifuged at 19,000g for 6 minutes at
4 C.
25 After the centrifugation the upper aqueous layer (600 l) was carefully
removed and
placed in a new tube. To the aqueous phase 650 1 of guanidium buffer (8M
guanidine
hydrochloride; 20mM EDTA; 20mM MES, pH 7.0) was added, then 1-
mercaptoethanol with final concentration of 200mM was added. The solution was
mixed well and then 350 1 of phenol: chloroform:isoamyl alcohol (25:24:1) was

30 added. The tube was then centrifuged at 19,000 g for 6 minutes at 4 C.
After the


CA 02510950 2005-06-14

51
centrifuge the upper aqueous phase was removed without disturbing the
interface. To
the upper aqueous phase 5001A1 of chloroform was added and mixed well. The
tube
was again centrifuged at 19,000g for 6 minutes at 4 C. The upper aqueous phase
containing the nucleic acids was carefully transferred to two new 2ml
microcentrifuge
tube (- 600 l each). To each tube, 601A1 of 3M sodium acetate (pH 5.2) and
1.2m1 of
chilled 100% ethanol was added. The tubers were inverted gently and then
incubated
at -75 C for 2 hours. After incubation the tubes were centrifuged at 19,000g
for 20
minutes at 4 C. The supernatant was discarded and the RNA pellet was washed
with
80% ethanol. The tube was then air dried at room temperature for 10 minutes.
The
RNA pellet was dissolved in 20 l of autoclaved deionised filter-sterilized
water. The
RNA was run on a 1% agarose gel to check for the quantity and quality.

DNase Treatment

The isolated RNA was treated by DNase-I (Promega Corp., WI, USA) to
remove any residual DNA contamination that may interfere with the RT-PCR
reactions. The RQ 1 RNAse free - DNase (Promega Corp., WI, USA) was used to
treat the isolated total RNA. The 401Al DNAse reaction mix contained the
following
l (-10 g) RNA, 4 l lOX DNAse buffer (400mM Tris-HCI, pH 8.0; 100mM
MgSO4; 10mM CaCI2), 25U RNAse inhibitor (Promega Corp., WI, USA), 15 I
RNAse free water. The mixture was mixed well and then incubated at 37 C for 1
20 hour. After the incubation the volume of the reaction mix was made up to
300 I using
RNAse free water. To the solution 30O I of phenol: chloroform:isoamyl alcohol
(25:24:1) was added and mixed well. The tube was centrifuged at 14,000g for 10
minutes. The upper aqueous phase was removed carefully to a new
microcentrifuge
tube. To the solution 200 l of chloroform was added, mixed well and
centrifuged for

10 minutes at 14,000g. The upper aqueous phase (-300 1) was removed and 30 l
of
3M sodium acetate (pH 5.2) and 600 l of chilled 100% ethanol was added. The
solution was mixed gently and then incubated at -75 C for 2 hours. After
incubation
the tube was centrifuged at 19,000g for 20 minutes at 4 C. The RNA pellet was
washed with 80% ethanol, air dried for lOminutes and dissolved in 10 l of
autoclaved


CA 02510950 2005-06-14

52
deionised filter-sterilized water. One microliter of the RNA sample was loaded
in a
1% agarose gel to estimate the concentration and test the quality of the total
RNA.
Reverse transcription

The single stranded eDNA synthesis from the total RNA was carried out using
avian myeloblastosis virus reverse transcriptase (AMV-RT) (Roche Applied
Science,
PQ, Canada). Random Primers (Roche Applied Science, PQ, Canada) were used to
reverse transcribe the RNA to single stranded cDNA. A 25 l reverse
transcription
reaction mix contains -650ng of total RNA, 20U of AMV-RT (Roche Applied
Science, PQ, Canada), 5 l of incubation buffer (50mM Tris-HCI; 8mM MgC12;

30mM KCI; 1mM dithiothreithol, pH 8.5), 5 l dNTPs (10mM), 2.5 1 of lOX random
hexanucleotides (Roche Applied Science, PQ, Canada), 25U of RNAse inhibitor
(Promega Corp., WI, USA) and ddH2O to a total volume of 25 l. The mixture was
incubated at 42 C for 70 minutes and the enzyme was deactivated at 80 C for 5
minutes.

Determination of linear range sensitivity for imaging device

The UVP Imaging device was used to quantify the differential gene
expression analysis. The linear range of sensitivity of the device has to be
determined
for absolute quantification of intensities of the PCR product bands in an
agarose gel.
This was determined by performing a series of PCR experiments with varying
initial
copy numbers of a vector plasmid. The vector plasmid used was a pGEMT vector
cloned with the C4H gene PCR product of about 514bp. The copynumbers of the
plasmid was estimated on the size and concentration of the plasmid
(Arumugananthan
and Earle, 1991). The initial copy numbers of the plasmid ranged from 102 to
1010
copies. A 27 cycle PCR was carried out and the PCR products were run on a 1.2%
agarose gel. The intensities of the bands were measured using the LabworksTM
software (UVP Inc., Upland, CA, USA). A graph was plotted for measured the
maximum pixel density of the bands against the initial copynumbers.


CA 02510950 2005-06-14

53
Optimization for relative quantitative RT-PCR

Accurate quantification of differential gene expression analysis using
relative
quantitative RT-PCR needs optimization of the following; 1) annealing
temperature
for selected gene specific primers, 2) PCR cycle numbers and 3) the internal
standard
for each selected candidate gene. All the PCRs for optimization and relative
quantitative RT-PCR were performed in Bio-Rad iCycler thermal cycler (Bio-Rad
Laboratories, ON, Canada).

1) Optimization of annealing temperature and PCR cycle number

The annealing temperature of the selected genes was first optimized by
performing RT-PCR for each gene with varying annealing temperatures. The
annealing temperatures tested were ranged from 50 C to 53 C.
In a PCR the amplified products tend to reach a plateau stage after reaching a
threshold cycle. It is therefore necessary to find the threshold cycle limit
for each
candidate gene before analyzing them together for differential gene expression
analysis. The threshold limit for the PCR cycle was determined by performing a
series
of PCR.

2) Optimization of internal standard

The 18s rRNA primers (QuantumRNATM, Ambion inc., TX, USA) can
amplify 315bp fragment specific to 18s rRNA in all plants. The 18s rRNA
primers
were used as an internal standard to monitor any sample to sample variation in
the
amount of initial cDNA. It also acts as an internal control for any
differences in the
reverse transcription and/or PCR processes. The 18s rRNA is abundant in the
isolated
total RNA which makes it difficult to be used as an internal control. The use
of
competimers overcomes this difficulty. The competimers are short sequences
homologous to 18s rRNA primers but their 3' end is blocked, therefore they
cannot be
amplified by the Taq polymerase. The competimers (homologous to 18s rRNA
primers) compete with the cDNA of 18s rRNA for binding with the 18s rRNA
primers. Thus, the use of competimers along with the 18s rRNA primers reduce
the
amplification of the 315bp fragment of 18sRNA PCR product during PCR.
Therefore


CA 02510950 2005-06-14

54
the ratio of the primer to competimer determines the amount of 315bp fragment
of
18sRNA during a PCR (Ambion inc. Tx, USA).
The internal standard 18s rRNA primers and competimers were mixed in
appropriate proportions (Table 9) to obtain the respective ratio. The optimum
primer
to competimer ratio to be used as an internal standard for each candidate gene
had to

be identified. This was determined by performing PCR for the gene with varying
18s
rRNA primers to competimer ratios (3:7, 2:8, 1:9). To each 20yc1 PCR reaction
tube,
1.6y l of the appropriate 18s rRNA primers to competimer ratio mix were added
and
PCR was performed at respective annealing temperatures. The PCR products were
run on a 1.2% agarose gel.

Relative quantitative RT - PCR

Single-stranded cDNA served as the template for relative quantitative RT-
PCR. The designed gene specific primers with optimized annealing temperatures,
PCR cycle numbers and 18s rRNA primers to competimers ratio were used to
determine the differential gene expression levels of C4H. Each relative
quantitative
RT-PCR mix contained one unit of Master Taq polymerase (Eppendorf, Brinkmann
instruments inc., Canada); 2 l of the single stranded cDNA template, 2 l of
PCR
reaction buffer (10X) containing 100mM Tris-HCI, pH 8.3; 15mM MgC12; 500mM
KCI; 1% Triton X-100; 2 l Taqmaster (5X PCR enhancer), 200 M of each dNTP;

0.5 M of upstream and downstream primers (specific for each selected candidate
gene); 1.6 1 of optimized 18s rRNA primers and competimers mix (Table 9) and
the
final volume was made up to 20 l. PCR was done with initial denaturation at 95
C for
2 minutes, followed by optimized cycles of denaturation at 95 C for 45
seconds,
annealing (temperature varies for each primer pair) for 45 seconds, extension
at 72 C
for 45 seconds and a final extension at 72 C for 7 minutes. The PCR product
was
examined on 1.2% agarose gel.

Quantification of differential gene expression

The relative quantitative RT-PCR products were run on an agarose gel and the
intensities of the bands were measured using the LabworksTM software (UVP
Inc.,
Upland, CA, USA). Maximum pixel intensities were measured for each relative


CA 02510950 2005-06-14

quantitative PCR reaction. There should be two bands present on a single lane,
one
band represents the amplified candidate gene specific fragment and other the
315bp
fragment of the internal standard (18s rRNA fragment). A blank reading of
maximum
pixel density with no band was also measured for each gel. The maximum pixel
5 density readings of both bands were taken and subtracted by the maximum
pixel
density of the blank. The absolute measurement of gene expression level was
calculated using Equation 1 given below.

10 Target gene pixel density - Blank
Gene expression level = -------------------------------------------------------
-X Internal standard ratio
Internal standard pixel density - Blank

Equation 1: Equation used to normalize the gene expression data obtained in
maximum pixel
15 intensities.

Measurement of chlorogenic acid and citric acid contents

Fresh potato tuber samples were used for measuring chlorogenic acid and
citric from tubers. Two repeated measurements for chlorogenic acid and citric
acid
20 were taken for each tuber sample.

1) Extraction of organic acids from tubers

The potato tubers were washed and peeled with a vegetable peeler to remove
all skin. Any bruising or rotten spots were removed using a small paring
knife. One
centimeter outer layer tissue was used and each tuber was chopped into small
pieces.
25 The chopped pieces were blend in a food processor for about 2 minutes.
Accurately
25.000g ( 0.001g) of the blended tuber was weighed in a beaker. To the blended
tuber, 50m1 of extract solution (70% methanol) was added and mixed for 5
minutes
on the magnetic mixer. The sample was then filtered through Whitman No.2
filter
paper using a Buckner filtration set up. The filter paper was washed with 50m1
of
30 70% methanol into the original beaker. The sample slurry was registered and
pooled
together with the previous filtrates. One milliliter of the extract was
pipette out into an


CA 02510950 2005-06-14

56
acid wash. The sample was then dried using nitrogen evaporator at 40 C. The
dry
sample was stored at -20 C until analyzed by HPLC.

2) HPLC analysis

The sample stored in -20 C was re-dissolved in lml of mobile phase (20mM
potassium phosphate buffer, pH 2.7) by overtaxing and brief sanitation. The
sample
was then filtered through a 0.22 m syringe filter. Twenty microliters of the
sample
was injected into the LKB (Bromma) HPLC. The HPLC was connected to a variable
wavelength detector and a spectra-physics SP4290 integrator.
For chlorogenic acid (CgA), the mobile phase was 15% acetonitrile in 20mM
potassium phosphate buffer (pH 2.7). The sample was run on an isocratic run at
0.75ml/minute for 20 minutes. The absorbance was measured at 325nm. For citric
acid (CA), the mobile phase was 20mM potassium phosphate buffer (pH 2.7), with
the flow rate of 0.75m1/minute on an isocratic run. The absorbance was
measured at
230nm.

Experimental Design

The experimental design of this study has two different sections: 1) for
differential gene expression analysis using relative quantitative RT-PCR and
2) for
chemical content measurements, such as chlorogenic acid (CgA), citric acid
(CA),
and chlorogenic acid to citric acid ratio (CgA:CA). A schematic representation
of the
experimental design is given in Figure 5 .

The RNA samples from the selected ACD dark or light samples of each
diploid families (13610-T and 13395-B) were pooled together for differential
gene
expression analysis using relative quantitative RT-PCR. Two separate reverse
transcription reactions for single-stranded cDNA synthesis were performed
using
pooled ACD dark or ACD light RNA samples individually for each diploid family
(13610-T and 13395-B). From each single-stranded cDNA sample obtained, two
relative quantitative PCR experiments were carried out. Therefore, there were
four
individual relative quantitative PCR experiments carried out. Similar
arrangement
was done for the differential gene expression of C4H in the tetraploid
cultivars


CA 02510950 2005-06-14

57
Shepody and Russet Burbank. All the four individual relative quantitative PCR
experiments were considered as four replications in this study.
Chlorogenic acid, citric acid and chlorogenic acid to citric acid measurements
for all the diploid families (13610-T and 13395-B) and tetraploid cultivars
(Shepody
and Russet Burbank) were determined twice, individually for each selected
tuber
sample. The CgA, CA and CgA:CA measurements of selected ACD dark or light
tubers were considered as replications for ACD dark or light clones in this
study.
Statistical Analysis

1) Differential gene expression analyses

The pixel density values obtained from four individual relative quantitative
RT-PCR analyses were normalized separately using the Equation 1. The
statistical
analyses of the normalized gene expression data was done in two ways. First
the fold
increase or decrease in the expression of the candidate in the dark clones
against the
light clones was analyzed using students t test. Secondly, significant
differences in the
candidate gene expressions among the dark and light clones of each family and
tetraploid cultivars were analyzed individually using one-way ANOVA. The four
individual PCR experiments carried out from two separately synthesized single-
stranded cDNAs were assumed as four replications of an experiment. The
statistical
analyses were carried out only for the data set that achieved normality. All
the
significant differences among the means were found at p< 0.05.

2) Statistical analyses on chemical analyses

Significant differences in the concentration of CgA, CA, CgA to CA ratio and
iron among the dark and light clones of each family and tetraploid cultivars
were
analyzed individually using one-way ANOVA. The concentration of CgA, CA, and
CgA to CA ratio obtained from each clone of dark or light were pooled together
for
each family. This pooled chemical data was assumed to be replicated values of
the
dark or light sample of that family. The statistical analyses were carried out
only for
the data set that achieved normality at p>0.1. All the significant differences
among
the means were found at p< 0.05.


CA 02510950 2005-06-14

58
RESULTS AND DISCUSSION

ACD Evaluation

Two diploid families, 13610-T and 13395-B, and two tetraploid cultivars,
Shepody and Russet Burbank, were evaluated for their ACD in Janurary, 2003 and
January, 2004. The ACD levels for the tubers were determined using the mean
pixel
density values obtained by Labworks digital imaging analysis software. ACD
light
tubers were determined by high pixel density values and ACD dark tubers
determined
by low pixel density values.
Many methods on evaluation of ACD in potato tubers have been reported,
they include the use of visual evaluation, high performance liquid
chromatography
(HPLC), gas chromatography, UV spectrophotometry and nitrous acid (Hughes et
al.,
1962; Chubey and Mazza, 1983; Siciliano et al., 1969; Griffiths et al., 1992).
The
ACD evaluation methods involving HPLC, gas chromatography are time consuming.
The visual evaluation of ACD, require proper standards to eliminate the
subjectivity
of the evaluator. Also some of the methods were unreliable as they analyze
only a
small portion of the tuber tissue. After-cooking darkening was evaluated using
digital-
imaging system in this study (Wang-Pruski and Tarn, 2003). The evaluation of
ACD
of potato tubers using the digital-imaging, in comparison to the earlier
methods is
fast, simple, accurate and consistent. The digital imaging analysis approach
allowed
the entire surface of the tuber to be analyzed; thereby any internal variation
was taken
into account. ACD evaluation was carried out using 2 tubers (4 halves) from
each
sample. The entire ACD evaluations for all the families, Shepody and Russet
Burbank
were performed by the same lab technician for the two years (2003 and 2004)
period,
which reduces potential manual error.
The distributions of the ACD among the clones of are shown in the Figure 6
and 7, respectively. The distribution of ACD was similar in family 13610-T and
13395B. In the family 13610-T, the pixel densities of the darkest clones were
82.07


CA 02510950 2005-06-14

59
and the pixel densities of the lightest clones were 134.48, respectively
(Table 10).
Family 13395-B did not show as wide good distribution pattern as 13610-T, but
segregated well for ACD. The pixel density of the darkest clone in family
13395-B
was 98.52 and the lightest clone had a pixel density reading of 132.41 (Table
10).

After-cooking darkening was evenly segregated among the progenies of the
diploid families in this study. The segregation data showed that diploid
family
13610-T had a more wide range of segregation (Table 10) than the 13395-B
family
(Table 10) (Wang-Pruski, unpublished).

Sample selection

The clones were selected based on their ACD values of both January 2003 and
January 2004. The ACD dark sample groups contained clones that showed very
high
ACD levels in both years; the ACD light group contained clones that showed
very
low ACD levels in both years. Three clones with the lowest or the highest
pixel
density readings in both January, 2003 and 2004, from the families 13610-T
were
selected (Table 11). Similarly two clones each with lowest and highest pixel
density
reading in both January 2003 and 2004 was selected in family 13395-B. The
pixel
density values of ACD dark and light clones selected from the families 13610-T
and
13395-B with their respective clone numbers are tabulated in Table 11. The two
tetraploid cultivars selected were Shepody and Russet Burbank in the same
table. The
ACD measurements were performed for the two cultivars as well.

Total RNA isolation

Total RNA was isolated from the frozen tuber samples based the protocol
given by Singh et al (2003). The quality of the isolated total RNA was tested
on a
1.0% agarose gel (Figure 8). Figure 7 shows the total RNA with two ribosomal
RNA
(rRNA) bands, one for 28s rRNA and another for 18s rRNA. The intensities of
both
the bands were in the ratio of 2:1, and there was no visible degradation of
RNA (Lane
1; Figure 8). The isolated total RNA was treated with DNase and extracted
using
phenol: chloroform as previously described. The DNAse treatment efficiently
removed the DNA contamination found in the total RNA isolation (Lane 2, 3;
Figure


CA 02510950 2005-06-14

8). The concentration of the isolated total RNA was calculated by the
LabworksTM
software using the known concentration of the ?-Hind III marker bands. About
20 to
25 g of total RNA was isolated from 300mg of ground potato tissue. The
isolated
total RNA was used for single-strand cDNA synthesis.

5 Single-stranded cDNA synthesis

Single-stranded cDNA was synthesized from the pooled total RNA from dark
or light samples of 13610-T and 13395-B by reverse transcription. The total
RNA
from the dark or light clones of the diploid families was pooled together in
single-
strand cDNA synthesis for use in differential gene expression analysis.
Pooling RNA
10 from similar samples have been proven to be more effective that using them
separately (Kendziorski et al., 2003; Xuejun et al., 2003). RNA samples are
pooled
together in microarray analyses to reduce the cost of the experiment and also
avoid
the biological variation (Kendziorski et al., 2003). Gene expression
measurement by
RT-PCR in individual samples shows that the variability in the measurements is
due
15 to the biological and technical variability. In case of RNA samples pooled
together
the variability observed in RT-PCR measurements are only due to experimental
variability (Kendziorski et al., 2003). Investigations on statistical
properties of RNA
pooling using data from real experiments and computer simulations, showed
appropriate pooling of biological samples is statistically valid and more
efficient for
20 microarray experiments (Xuejun et al., 2003).
The pooled total RNA samples of dark or light samples of diploid families and
total RNA from Shepody and Russet Burbank were reverse transcribed to single-
stranded cDNA using random primers. Random primers are used as AMV reverse
transcriptase can reverse transcribe both rRNA and messenger RNA (mRNA). The
25 reverse transcribed rRNA was required for the use of 18s rRNA primers which
were
used as an internal standard for relative-quantitative RT-PCR. The quality of
the
synthesized single-stranded cDNA was determined by visualization on 1.0%
agarose
gel using gel electrophoresis (Figure 11). The quality appeared to be
satisfactory as it
had an even smear between 5000bp to 500bp. The cDNA samples were quantified by
30 the calculating the pixel density area of the cDNA smear on the gel against
the X-
Hind III marker bands using LabworksTM software. The differential analyses of


CA 02510950 2005-06-14

61
candidate genes were carried out using ACD dark or light single-stranded cDNA
with
appropriate candidate gene specific primers and internal standards.

Determination C4H specific primers

The C4H specific primers were designed using Primer3 software for the
nucleotide sequences of the candidate gene cinnamic-4 hydroxylase (C4H). The
full
length cDNA sequences from potato used for designing primers (Table 13). The
gene
specific primers target the 514bp fragment of C4H. The nucleotide sequence of
the
chosen set of primers for the C4H gene is given in Table 13. These gene
specific
forward and reverse primers were used in relative quantitative RT-PCR for
determining the differential gene expression analysis.

Optimization for relative quantitative RT-PCR

Relative quantitative RT-PCR is a semi-quantitative, medium throughput
technique for differential gene expression analysis on a small scale. The
relative
quantitative RT-PCR technique was selected in this study for its simplicity
and
reproducibility compared to other differential gene expression analysis
methods such
as Northern hybridization, competitive RT-PCR, Real-time RT-PCR. One major
issue
of relative quantitative RT-PCR is that several optimizations have to be
performed for
obtaining reproducible and valid results. Some of the parameters to be
optimized for
relative quantitiative RT-PCR are: to determine the linear range sensitivity
of the
imaging device, optimal annealing and PCR cycle numbers and use of an
optimized
internal standard (18s rRNA primers to competimers ratio in this study).

1) Linear range sensitivity for imaging device

The threshold limit of the imaging device to differentiate between two white
pixels is determined by its linear range sensitivity. A PCR with increasing
copy
numbers of the pGEMT vector containing C4H PCR product was performed. The
maximum pixel densities of the amplified bands were measured using the UVP
imaging device (Figure 10). A graph (Figure 11) was plotted for intensities of
the
bands in Figure 10 against the initial copy numbers of the plasmid. The graph
shows
an initial phase followed by the exponential phase and finally a plateau
phase. The lag


CA 02510950 2005-06-14

62
phase shows the minimum level of sensitivity of the imaging device to identify
the
band intensity. The plateau phase shows the maximum level of sensitivity of
the
imaging device. The linear range of sensitivity of the imaging device is the
exponential phase was between 6 pixels to 215 pixels (Figure 11).

2) Annealing temperature and PCR cycle number

The optimum annealing temperature at which the designed primers can
efficiently bind to the target fragment was determined. The annealing
temperature for
C4H specific primers was 53 C.
The threshold limit for the PCR cycle was determined by stopping the PCR at
various cycles ranging from 23 to 29. Figure 12 shows the amplified PCR
products at
different cycles. The maximum pixel densities of the amplified products were
measured and the cycle number 27 was found to be optimum for all the candidate
genes tested. The intensity of the amplified DNA product at cycle 27 (178.0
maximum pixel density) was within the linear range of the sensitivity of the
imaging
device.

3) Optimization of 18s rRNA internal standard

The optimal primer to competimer ratio for each candidate gene, to be used as
an internal standard was determined. Polymerase chain reactions for the
candidate
gene (C4H) with the 18s rRNA primer to competimer ratio (1:9) was carried out
at
respective optimal annealing temperatures. The bands were visualized using UVP
imaging device to determine the intensity of the two bands on a single lane.
The first
band represented the PCR product of the target gene with respective fragment
size
and the other represented the 315bp PCR product of the 18s rRNA internal
standard.
The ratios of the intensities of both the bands were measured
The relative quantitative RT-PCR uses 18s rRNA as an internal control for
normalizing the gene expression data obtained. The 18s rRNA internal control
helped
in normalization of gene expression that accounted for any tube to tube
variation
caused by variable RNA or cDNA quality, inaccurate quantitation or pipetting.
The
use of 18s rRNA primers and competimers from Ambion gave reproducible results
once the ratios were optimized for different genes. Competimers along with the
18s


CA 02510950 2005-06-14

63
rRNA primers increased the possibility of identifying differential gene
expression
profiles of extremely rare mRNA transcripts. The 18srRNA primers to competimer
ratio helped in determining the true gene expression of the candidate genes
using the
Equation 1.
The Equation 1 was used to normalize the gene expression levels based on the
18s rRNA internal control. The normalized gene expression values obtained
using
Equation 1 can be directly correlated to the level of gene expression in the
samples.
The pixel density values of the internal controls for the dark or light
samples of
respective candidate gene in a PCR experiment was similar. This validates the
efficiency of the internal control used.
The optimization of the annealing temperatures for the designed gene specific
primers, PCR cycle numbers for differential expression of candidate genes,
linear
range sensitivity of the imaging device and the 18s rRNA primer to competimer
ratios
of the internal standard were important for differential gene expression
analysis using
relative quantitative RT-PCR. These optimizations validate the results
obtained, and
account for reproducibility. Using these optimizations, the differential gene
expression analysis was performed on the selected dark and light clones of
diploid
families and the tetraploid cultivars.

Differential expression of C4H gene and its relationship to ACD

In this study, Families 13610-T, 13395-B and Russet Burbank/Shepody were
selected as a model to study C4H gene expression and its relationship to ACD.
This
section focuses on the results and discussion of ACD data analysis, chemical
content
analysis and differential expression of C4H for the family 13610-T. The
relationship
of chemicals such as CgA, CA, and CgA to CA ratio are correlated to ACD. The
differential expression of C4H gene is correlated to its respective chemical
products
and finally to ACD.

1) ACD data analysis

The ACD values of the selected dark and light clones of the families of 13610-
T and 13395 were measured as mentioned above.


CA 02510950 2005-06-14

64
2) Chemical data analysis

The concentration of CgA and CA concentration in the selected dark and light
clones of families of 13610-T and 13395-B were measured using HPLC. The mean
concentrations of CgA, CA, and CgA to CA ratio in the selected tuber clones
are
tabulated in Table 15. The concentration of CgA was higher in the dark clones
of
family 13610-T than that of the light clones. The CA concentration in the
light clones
of family 13610-T was higher than that of the dark clones. The CgA to CA ratio
also
was found to be higher in the dark clones of 13610-T compared to the light
clones.

One way ANOVA analyses was performed on the CgA, CA contents, and
CgA to CA ratio in the dark and light clones separately (Table 15). The
results show
that the CgA in the dark clones of family 13610-T was significantly higher
than that
of the light clones (P = 0.046). In family 13610-T, the mean CgA content in
the dark
clones was 0.49 mg 100-'g and the mean CgA content in the light clones was
0.24 mg
100-'g. The statistical analysis of CA concentration among the dark and light
clones
of family 13610-T are tabulated in Table 15.

The results show that the CA content in the dark and the light clones are not
significantly different (P = 0.617). The mean CA concentration of the dark
clones and
the light clones of family 13610-T are 808.15 mg 100"'g and 833.47mg 100"'g.
The
statistical results of CgA to CA ratio are given in Table 15. The results show
that the
CgA to CA ratio in the dark clones of family 13610-T was significantly higher
than
that of the light clones (P = 0.049). The mean CgA to CA ratio of the dark
clones and
the light clones were 6.05x10-4 and 2.95x10-4, respectively. Similar results
were also
reported by Hughes and Swain (1962 b). Hughes and Swain (1962 b) from their in-

vitro experiments on CgA, CA and CgA:CA found that CgA and CgA to CA ratio to
play an important role in ACD. They also found no significant changes in the
citric
acid levels among the tubers they analyzed.

3) Relative quantitative RT-PCR

Relative quantitative RT-PCR was performed for the pooled ACD dark or
light clones of the families 13610-T, 13395-B and Russet Burbank and Shpody.
The
relative quantitative RT-PCR was done separately for single-stranded cDNA from
dark or light samples. Four separate PCR experiments (PCR1, PCR2, PCR3 and


CA 02510950 2005-06-14

PCR4) were carried out for each gene from two separately synthesized single-
stranded cDNA of dark or light samples. All the PCR experiments were carried
out
for 27 cycles.
The relative quantitative RT-PCR results obtained for the four PCR
5 experiments in the following Figures. Each relative quantitative RT-PCR
shows two
bands: the amplification of the gene specific band of the candidate gene and
the
315bp internal standard amplified by the 18s rRNA primers. The amplification
of the
internal standard differs according to the ratio of the 18s rRNA primer and
competimer used. The maximum pixel densities (MPD) for the two bands and a

10 blank reading were measured. The normalized gene expression level for each
PCR
was calculated using Equation 1. The maximum pixel densities and the
normalized
gene expression are illustrated in Tables below. The graphical representation
of the
normalized gene expression levels of the C4H are shown in Figures below.

4) Differential cinnamic acid 4-hydroxylase gene expression

15 The differential expressions of cinnamic acid 4-hydroxylase (C4H) gene
between dark and light samples of family 13610-T is shown in Figures 13 and
14. It
contains the gel pictures of four PCR experiments. The C4H gene specific PCR
product of about 514bp was detected consistently in all the four PCR
experiments
along with the 315bp internal standard. The internal standard ratio of the 18s
rRNA
20 primers and competimers used for C4H gene differential analysis is 1:9. The
maximum pixel densities (MPD) of the bands are given in Table 16. The table
shows
the normalized gene expression levels of C4H gene calculated using the
Equation 1.
The normalized gene expression values of the dark clones are consistently
higher than
that of the light clones in the family 13610-T (Figure 14).
25 The C4H gene expression in the dark clones of family 13610-T was about 6
fold higher than that of the light samples, when the means of the normalized
C4H
gene expressions between dark and light clones were compared (Figure 19). The
four
repeated PCR experiments showed a consistently higher expression of C4H gene
expression in the dark samples compared to that of the light samples (Figure
14). The
30 one way ANOVA results on the normalized C4H gene expression levels of dark
and
light samples of family 13610-T is shown in Figure 20. The results of the one-
way


CA 02510950 2005-06-14

66
ANOVA analysis on C4H gene expression showed that the dark samples of family
13610-T is significantly higher than that of the light samples (P = 0.021).
The highest
mean normalized C4H gene expression was 0.36 for the dark samples and the
lowest
mean normalized C4H gene expression was 0.08 in the light samples (Figure 20).
The C4H gene expression in the ACD dark clones of 13610-T was about 6
fold higher than the light clones (Figure 19). This shows that in family 13610-
T,
ACD dark clones had high level of C4H gene expression and the ACD light clones
of
family 13610-T had low level of C4H gene expression. It was also be noted that
the
ACD levels between the dark and light clones were significantly apart in
family
13610-T (Table 10). The normalized gene expression values of C4H gene were
always higher in the ACD dark clones than that of the ACD light clones (Table
16).
Therefore, it could be concluded that the C4H gene expression is always higher
in the
ACD dark clones and the expression of C4H gene was lower in the ACD light
clones
in this study.
One-way ANOVA was performed to study the significant differences between
the C4H gene expression between the ACD dark and light clones (Figure 20). The
statistical analysis showed that the C4H gene expressions in the ACD dark
clones of
family 13610-T was significantly higher than that of the light clones (P =
0.021). This
shows that C4H gene was highly expressed in ACD dark clones of family 13610-T
and the C4H gene expression in the ACD light clones was considerably lower.
This
correlates with the 6 fold increase in the C4H gene expression in the ACD dark
clones
of family 13610-T than that of the ACD light clones (Figure 19). Cantle (2004)
performed Northern hybridization analysis to determine C4H gene expression
between ACD dark and light clones. She reported that ACD dark clone had higher
C4H gene expression than that of the ACD light clone. Therefore it is evident
that the
C4H gene expression is significantly higher in the tubers with high ACD levels
and
the C4H gene expression is lower in tubers with low ACD levels.
The CgA and CgA to CA concentration in the dark clones of family 13610-T
was also significantly higher than that of the light clones (Table 15). This
shows that
CgA and CgA to CA ratio are high in the dark clones compared to the light
clones.
Similar results were reported by Hughes and Swain (1962 a, b). It is noted
that the
significant increase in the expression of C4H is followed by a significant
increase in


CA 02510950 2005-06-14

67
CgA in dark clones of family 13610-T. This shows that there is a strong
correlation
between the expression of C4H and CgA concentration in the tubers. C4H enzyme
being an important enzyme in the phenylpropanoid pathway could possibly play
an
important role in the synthesis of CgA.

In this study, the relative quantitative RT-PCR data strongly correlated with
the C4H gene expression to ACD at different levels. The fold increase of C4H
gene
expression also correlated with the difference in the ACD dark and light
clones in the
diploid families. C4H gene expression correlated with the CgA concentrations
in the
samples studies. From these observations, it can be concluded that C4H gene is
a
potential gene for regulating ACD levels in potato tubers.

Very similar data have been obtained from 13395-B and the two tetraploid
samples (Tables 14, 15, 17, 18 and Figures 15, 16, 17, 18, 19, 20, 21). The
data from
samples for family 13395-B and the tetraploid cultivars have shown very
similar gene
expression patterns as 13610-T; the chemical contents of these samples also
correlate
with ACD in these samples.

Family 13610-T was selected as a model to study the relationship of candidate
gene expressions and chemical contents to ACD. The ACD values of the dark and
light clones selected were significant to provide enough information on ACD.
The
findings from this study support the hypothesis that CgA is the main chemical
involved in ACD. Also the chlorogenic to CA ratio was found to play a major
role in
ACD. The CA and iron contents did not show any relationship to ACD. These
results
support the findings of Hughes and Swain (1962 a, b), Swiniarski (1968), Wang-
Pruski et al. (2003). This confirms that the selected dark and light clones of
family
13610-T served as a good model for studying ACD trait. Therefore, the use of
these
selected clones to study the differential expression of candidate genes will
be
appropriate.

This is the first study to report the differential expression of candidate
genes
and their respective chemical product for their relationship to ACD.

The differential gene expression studies proved our hypothesis that the
expression of C4H gene would be high in the clones with increased CgA levels.
Our
differential gene expression analysis results supported the findings of
Landschutze et


CA 02510950 2005-06-14

68
at (1995), Ma et at. (2001), Petit et al. (2002), Cantle (2004), Topley
(2004),
Niggeweg et al. (2004). This showed that the differential gene expression
analysis
using relative quantitative RT-PCR is an efficient tool for differential gene
expression. The differential gene expression analysis indicated that C4H genes
could
be considered as potential gene candidates for ACD trait analysis.

Example 3
Real time PCR protocol:
= lul of gene specific frd. primer and rev. primer was added to 25u1 of
platinum
SYBR green qPCR super mix with ROX. The volume was made up to 48u1
with water.
= lul of C4H gene standard was added to 24u1 of the PCR mix and the real-time
PCR was carried out.
= Reaction conditions:
Cycle 1: ( 1X)
Step 1: 50.0 Cfor 02:00
Cycle 2: ( 1X)
Step 1: 95.0 Cfor 02:00
Cycle 3: ( 35X)
Step 1: 95.0 Cfor 00:15
Step 2: 53.0 Cfor 00:30
Step 3: 72.0 Cfor 00:30
Data collection and real-time analysis enabled.
Cycle 4: ( 1X)
Step 1: 72.0 Cfor 05:00
Cycle 5: (250X)
Step 1: 95.0 Cfor 00:15
Decrease set point temperature after cycle 2 by 0.1 C
Results:

Real time PCR raw data for 13610-T


CA 02510950 2005-06-14

69
C4H

Dark Light
PCR 1 1.65E+02 1.57E+02
PCR 2 1.92E+02 1.69E+02
PCR 3 1.57E+02 1.17E+02
PCR 4 1.76E+02 1.89E+02
Average 172.50 158.00
Normalized gene expression values
C4H
Dark Light
PCR 1 3.25 1.89
PCR 2 3.78 2.04
PCR 3 3.58 1.09
PCR 4 4.02 1.75
Average 3.66 1.69

The above real time PCR results strongly supported the finding using relative
quatatative RT-PCR

Example 4: Using RNAi technique to inhibit C4H gene expression in potato
Background information regarding RNAi can be fund from:
Martin Hartmut Schattat, Ralf Bernd Klosgen and Joao Pedro Maroues. A Novel
Vector for Efficient Gene Silencing in Plants. Plant Molecular Biology
Reporter,
2004, 22: 145-153.

P. Susi, M. Hohkuri, T. Wahlroos and N.J. Kilby. Characteristics of RNA
silencing in
plants: similarities and differences across kingdoms. Plant Molecular Biology,
2004,
00: 1-18.


CA 02510950 2005-06-14

Neema Agrawal, P. V. N. Dasaradhi, Asif Mohmmed, Pawan Malhotra,
Raj K. Bhatnagar, and Sunil K. Mukherjee. RNA Interference: Biology,
Mechanism,
and Applications. Microbiology and Molecular Biology Reviews, 2003, 67(4):
657-685.
5
Derek M. dykxhoorn, Carl D. Novina and Phillip A. Sharp. Killing the
messenger:
Short RNAs that silence gene expression. Nature, 2003, 4: 457-467.

Inhibition of C4H gene expression in plants have been carried out in the
10 following two steps.

1. Generation of C4H silencing cassette
The pJM007 (Schattat, et al., 2004, Plant Molecular Biology Reporter 22:
145-153) vector system is used for this study. It contains the second intron
(IV2) of
15 the ST-LS 1 gene from potato. The full length and partial gene of C41-1
(the partial
sequence of C4H, around 500 bp, comes form the 5' end of the gene) are used to
generate two separate constructs. The sequence in sense orientation was
ligated into
the pJM007 at the right side of the ST-LS 1 intron and the antisenes on the
right side.
Therefore, the developed C4H RNAi cassette contains the sense sequence of C4H,
20 ST-LS I intron and the antisense sequence of C4H in order to produce the
hairpin
RNA with the intron in the middle as the loop, which will trigger the specific
gene
inhibition.

2. Plant transformation (method listed previously)
The above silencing cassettes are to be excised from pJM007 and cloned into
the
binary vector pGreenII0129. Electroporation is used to transfer the resulting
constructs to Agrobacterium tumefaciens GV3101 (pMP90). Potato explants (var
Bintje) are transformed under the Agrobacterium mediation and selected on CSM
(callus selective medium) containing 501tg/ml of kanamycin. The transformed
plants
will be verified using PCR, Southern hybridization and antisense expression
will eb
verified using RT-PCR. Antisense activity will be further assessed and the
biological
assays will follow. Chlorogenic acid will be evaluated and ACD in potato tuber
will
be measured as well.


CA 02510950 2005-06-14

71
Example 5
Up-reulation of C4H gene expression in potato
The full length C4H gene will be subcloned into the plant expression vector
pGreenII0l29 containing the tCUP promoter will be transferred into potato
plantlets.
The aim of this study is to over produce chlorogenic acid which is recognized
as a
natural occurring antioxidant.
15
25


CA 02510950 2006-09-14

72
TABLE 1: Sequences of designed primers for cloning of c4h by PCR*

Primer ID SEQ ID NO Primer Sequence (5' to 3') Tm ( C)
AF AR 7 5'-CCCCAGGTCCAATTCCA-3' 60.25
8 5'-TTCAGGGGATGACACAACAG-3' 59.52
BF BR 9 5'-CTGTTGTGTCATCCCCTGAA-3' 59.52
5'-CCTCATTTTCCTCCAGTGCT-3' 59.28
CF CR 11 5'-GGCCTTTCTTGAGGGGTTAC-3' 59.94
12 5' -CCTCGTTGATCTCTCCCTTCT-3' 59.83
DF DR 13 5'-GAAGGGAGAGATCAACGAGG-3' 58.82
14 5'-TCACAGCCTGAAGGTATGG-3' 57.16
EF ER 15 5'-CCACTGGAAGAAACCTGAAG-3' 57.36
16 5'-TTCTGCACCAAACGTCC-3' 56.43
FF FR 17 5'-AGCATTGGAGGAAGATGAGG-3' 59.24
18 5'-GCCAATCTACTCCTCTCAGCA-3' 59.59
GF GR 19 5'-GGCTTTGAATGGTGAGAGGA-3' 60.20
5'-TGGATATGAGGGTGGTTGAC-3' 58.20
* The first letter refers to the primer name (A to F) and the second letter
indicates the
direction of the primer, forward or reverse (e.g. AF AR).

TABLE 2:Sequences of primers for cloning c4h by 5' and 3' RACE*
Primer ID SEQ ID NO Primer Sequence (5' to 3') Tm ( C)
SPI 21 5'-TTCCTCCAGTGCTCACCATAC-3' 60.13
SP2 22 5'-GGTATAGAACTGGGAAGGGACA-3' 59.35
SP3 23 5'-CAGGGGATGACACAACAACT-3' 58.41
SP4 24 5'-AGAGGAGAAGCACGTTGAGG-3' 59.60
Oligo 25 5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3' n/a
dT-Anchor V= A, C, or G
PCR Anchor 26 5'-GACCACGCGTATCGATGTCGAC-3' 67.13
* Primers SP1, SP2, SP3, and SP4 were designed based on the c4h gene sequence
in potato. The
remaining primers were supplied with the 5'/3' RACE kit. Abbreviation: SP -
sequence specific
primer.


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73
TABLE 3: Sequence of the c4h gene in potato (SEQ ID NO: I)

1 AAACATTCTT TTCTCAAACT TCCCTCTGAA AGAACTCACC AAAAATGGAT 5'-UTR
51 CTTCTCTTAC TGGAGAAGAC CTTAATAGGT CTTTTCTTTG CTATTTTAAT
101 CGCTATTATT GTCTCTAAAC TTCGTTCCAA GCGATTTAAA CTACCCCCAG
151 GTCCAATTCC AGTCCCAGTT TTTGGAAATT GGCTTCAAGT TGGTGATGAT
201 TTGAACCATA GAAACCTTAC TGATTATGCT AAAAAGTTTG GTGATGTGTT
251 CTTGCTTAGA ATGGGGCAAA GGAACTTAGT TGTTGTGTCA TCCCCTGAAT
301 TAGCTAAATA AGTTTTACAC ACACAAGGGG TTGAATTTGG TTCAAGGACA
351 AGAAATGTTG TTTTTGATAT TTTTACAGGG AAGGGTCAAG ATATGGTTTT
401 TACAGTGTAT GGTGAGCACT GGAGGAAAAT GTGGAGGATT ATGACTGTAC Exon 1
451 CCTTTTTTAC TAATAAGGTG GTGCAGCAGT ATAGAGGGGG GTGGGAGTCT
501 GAGGCTGCTA GTGTAGTTGA GGATGTGAAG AAAAACCCTG AATCTGCTAC
551 AAATGGGATT GTTTTGAGGA AAAGATTGCA GCTTATGATG TATAATAACA
601 TGTTTAGGAT TAAGTTTGAT AAGAACTTTG AGAGTGAAGA TAATCCCCTT
651 TTTGTTAAGC TTAGGGCTTT GAATGGTGAG AGGAGTAGAT TGGCTCAGAG
701 CTTTGAGTAC AACTATGGTG ATTTTATCCC TATTTTGAGG CCTTTCTTGA
751 GAGGGTACTT GAAGATTTGT AAGGAGGT A AGGAGAAGAG GTTGAAGCTA
801 TTCAAAGACT ACTTTGTTGA TGAAAGAAAG TAAGTTCACT TTTTTCTTGT
851 TAATCCCTTT ATGCTCAATT TGATCATTTG TATCAGTTTT ATTTATTAGT
901 TTAGTTTAGT TGTAAGGGGT GTTTGACTAA ATCTTGGAAC AGTATGGATC
951 AATTTTGAAT AGAAAAGGAA GTACTAGTTG ACATTTCAGA ATAGTAAGGG
1001 TCCATTGGTT AAATTTTAAA AAAGGTAGTT CTTGTTTTCT GTTTTCAAAG
1051 TGATAATGAA AATTAGCGTG GTGTTTGGCA TATTTGGAGT TGTTTTGCGA Intron 1
1101 TTCTCCTGTG GCAATTAGAG GTTTGTCGTA ACGGTGGCCT GTGAGAGCCT
1151 AGCTTGCAGT GGTAAGAGTA GTGAGTGATT TGGAGTAAAA AAGTTAATAA
1201 CTTTTTGATT GATGTTTTTT AAATTTTTAG TTGAATTCCA GAATTGGCCA
1251 ATAAGAATCA TGTATGATTT AGTGATAGTT AAAGTGCTTT GAGGTACTGT
1301 TAGGTAGCTT TTGATGGTGG ACCTTGTGTT TTAGTTTGTA ATATTTTTAT
1351 TGCTTTACAC AGGAACCTTG CACACACCAA GAGCATGGAC AGCAATGCTC
1401 TAAAATGTGC AATTGATCAC ATCCTGGAAG CTCAACAGAA GGGAGAGATC Exon 2
1451 AACGAGGATA ACGTTCTTTA CATCGTTGAG AACATCAATG TTGCTGGTAT
1501 GTTTCGAAAT AACATATCTT TGATTCTCTA GAGTAAAATT TGTTCTAGTT
1551 TGGTTTAAAT GATTGCATCC TAGTTAGAAT AAAAGTAATT TATAAGTGAA
1601 TGAAAATCCA ATTCCAATTT TGTCTATTTT TCTCAAAAGT AGTAGTTGAG
1651 AGTTACCAAA TAAGGGGCCC AAGATTTAAC TGTTTTTTAT GTTGCCAAGG
1701 ACTAGTTGGT GCCTGGGCCC TGGGGGGTAC CACACACCAA TTTCTTGTGG
1751 TAAATAAGAT GTTATGTTTA CATCCAAGGA AGACATGTAG TTTCCAAGTT
1801 TGAAGGGGAA ATAAGTACTA TAGTAAAATG AACCACATGT TTCAAGTGAT
1851 GGCGATGTTT CTAGGCTAGG TTACAAAGAC TTGTTAGGTA CCACAATTCT Intron 2
1901 TATACTACTA TAAGACTTAA GTCCCAAACA AAGTTGGATA CAATCGGGTT
1951 CTATGGGTTT TACTGAATTC ATTGCTTTTG AAGTGTGCAT ACATATGAAA
2001 AAGAATTTGT AATGTATACA TATGTAATGA GATCATACAT ATTTTGAACT
2051 CAATAACGGG TAGATCTTGG AATTGCCTCT TGTCCGGAAG TTGTTTCATT
2101 TATTGCATCG CCTTGTAGTA AGTAATACAT GAGTTTTGAT ATGGTCTTAA
2151 ACTTAAAAAG TCACACATCC TACCATTGAA GCATGTTTTG TTGTTTATAT
2201 CTGTTCGTAA ACTTCTTGGT TAGTTGATTA TTCAGCTGAT ATGCTTAATT
2251 ACTGTCGTGA CCACCATTCG AAACAACATT GTGGTCAATT GAGTGGGGTA
2301 TCGCGGAACT AGTCAACCAC CCTCATATCC AAAAGTAATT CCGTGATGAG
2351 ATTGATACAG TTCTTGGACC AGGAATGCAA GTGACTGAGC CAGACATGCC
2401 CAAGCTTCCG TACCTTCAGG CTGTGATCAA GGAGACTCTT AGACTCAGGA
2451 TGGCAATTCC TCTTTTAGTC CCACACATGA ACCTTCATGA TGCAAAGCTT
2501 GCTGGATACG ATATTCCAGC TGAAAGCAAA ATCTTAGTTA ACCCATGGTG Exon3
2551 GCTAGCTAAC AACCCCGCTC ACTGGAAGAA ACCTGAAGAG TTCAGACCTG
2601 AGAGGTTCTT CGAAGAGGAG AAGCACGTTG AGGCCAATGG CAACGACTTC
2651 AGATTTCTTC CTTTCGGTGT TGGTAGGAGG AGTTGCCCCG GAATTATCCT
2701 TGCATTGCCA ATTCTCGGCA TCACTTTGGG AAGTTTGGTG CAGAACTTTG
2751 AGATGTTGCC TCCTCCAGGA CAGTCAAAGC TCGACACCTC GGAGAAAGGT
2801 GGACAGTTCA GTCTCCACAT TTTGAAGCAT TCCACCATTG TGATGAAACC
2851 AAGATCTAAC TAAACTTTGT AATGCTATCA ATTAATCATG ATTGTTGTTT 3'-UTR
2901 GTTTGTGTAA ACCTTTTAAG TTTGACAGAA AACATTCTTC TTTCTTATGT
2951 TTTATAAAAG TCTTATTGGA CTAGATTATT CATTAT


CA 02510950 2005-06-14

74
Table 4: The c4h coding sequence and predicted amino acid sequence in potato
1 ATG GAT CTT CTC TTA CTG GAG AAG ACC TTA ATA GGT CTT TTC TTT GCT ATT
1 M D L L L L E K T L I G L F F A I
52 TTA ATC GCT ATT ATT GTC TCT AAA CTT CGT TCC AAG CGA TTT AAA CTA CCC
18 L I A I I V S K L R S K R F K L P
103 CCA GGT CCA ATT CCA GTC CCA GTT TTT GGA AAT TGG CTT CAA GTT GGT GAT
35 P G P I P V P V F G N W L Q V G D
154 GAT TTG AAC CAT AGA AAC CTT ACT GAG TAT GCT AAA AAG TTT GGT GAT GTG
52 D L N H R N L T E Y A K K F G D V
205 TTC TTG CTT AGA ATG GGG CAA AGG AAC TTA GTT GTT GTG TCA TCC CCT GAA
69 F L L R M G Q R N L V V V S S P E
256 TTA GCT AAA GAA GTT TTA CAC ACA CAA GGG GTT GAA TTT GGT TCA AGA ACA
86 L A K E V L H T Q G V E F G S R T
307 AGA AAT GTT GTT TTT GAT ATT TTT ACA GGG AAG GGT CAA GAT ATG GTT TTT
103 R N V V F D I F T G K G Q D M V F
358 ACA GTG TAT GGT GAG CAC TGG AGG AAA ATG AGG AGG ATT ATG ACT GTA CCC
120 T V Y G E H W R K M R R I M T V P
409 TTT TTT ACT AAT AAG GTG GTG CAG CAG TAT AGA GGG GGG TGG GAG TCT GAG
137 F F T N K V V Q Q Y R G G W E S E
460 GCT GCT AGT GTA GTT GAG GAT GTG AAG AAA AAC CCT GAA TCT GCT ACA AAT
154 A A S V V E D V K K N P E S A T N
511 GGG ATT GTT TTG AGG AAA AGA TTG CAG CTT ATG ATG TAT AAT AAC ATG TTT
171 G I V L R K R L Q L M M Y N N M F
562 AGG ATT ATG TTT GAT AGG AGA TTT GAG AGT GAA GAT GAT CCC CTT TTT GTT
188 R I M F D R R F E S E D D P L F V
613 AAG CTT AGG GCT TTG AAT GGT GAG AGG AGT AGA TTG GCT CAG AGC TTT GAG
205 K L R A L N G E R S R L A Q S F E
664 TAC AAC TAT GGT GAT TTT ATC CCT ATT TTG AGG CCT TTC TTG AGA GGG TAC
222 Y N Y G D F I P I L R P F L R G Y
715 TTG AAG ATT TGT AAG GAG GTT AAG GAG AAG AGG TTG AAG CTA TTC AAA GAC
239 L K I C K E V K E K R L K L F K D
766 TAC TTT GTT GAT GAA AGA AAA AAG CTT GCA AAT ACC AAG AGC ATG GAC AGC
256 Y F V D E R K K L A N T K S M D S
817 AAT GCT CTA AAA TGT GCA ATT GAT CAC ATT CTT GAA GCT CAA CAG AAG GGA
273 N A L K C A I D H I L E A Q Q K G
868 GAG ATC AAC GAG GAT AAC GTT CTT TAC ATC GTT GAG AAC ATC AAT GTT GCT
290 I N E D N V L Y I V E N I N V A
919 G A ATC GAA ACA ACA TTG TGG TCA ATT GAG TGG GGT ATC GCG GAA CTA GTC
307 A I E T T L W S I E W G I A E L V
970 AAC CAC CCT CAT ATC CAA AAG AAA CTC CGT GAT GAG ATT GAT ACA GTT CTT
324 N H P H I Q K K L R D E I D T V L
1021 GGA CCA GGA ATG CAA GTG ACT GAG CCA GAC ATG CCC AAG CTT CCG TAC CTT
341 G P G M Q V T E P D M P K L P Y L
1072 CAG GCT GTG ATC AAG GAG ACT CTT AGA CTC AGG ATG GCA ATT CCT CTT TTA
358 Q A V I K E T L R L R M A I P L L
1123 GTC CCA CAC ATG AAC CTT CAT GAT GCA AAG CTT GCT GGA TAC GAT ATT CCA
375 V P H M N L H D A K L A G Y D I P
1174 GCT GAA AGC AAA ATC TTA GTT AAC GCT TGG TGG CTA GCT AAC AAC CCC GCT
392 A E S K I L V N A W W L A N N P A
1225 CAC TGG AAG AAA CCT GAA GAG TTC AGA CCT GAG AGG TTC TTC GAA GAG GAG
409 H W K K P E E F R P E R F F E E E
1276 AAG CAC GTT GAG GCC AAT GGC AAC GAC TTC AGA TTT CTT CCT TTC GGT GTT
426 K H V E A N G N D F R F L P F G V
1327 GGT AGG AGG AGT TGC CCC GGA ATT ATC CTT GCA TTG CCA ATT CTC GGC ATC
443 G R R S C P G I I L A L P I L G I
1378 ACT TTG GGA CGT TTG GTG CAG AAC TTT GAG ATG TTG CCT CCT CCA GGA CAG
460 T L G R L V Q N F E M L P P P G Q
1429 TCA AAG CTC GAC ACC TCG GAG AAA GGT GGA CAG TTC AGT CTC CAC ATT TTG
477 S K L D T S E K G G Q F S L H I L
1480 AAG CAT TCC ACC ATT GTG ATG AAA CCA AGA TCT TTC TAA
494 K H S T I V M K P R S F


CA 02510950 2005-06-14

TABLE 5: Mean relative intensity of c4h expression as detected by Northern
hybridization analyses*
Tuber Sample Number of Mean Relative Intensity
Observations of c4h Expression
Dark Diploid (10908.06) 3 1.70 */- 0.53 a
Russet Burbank 3 1.12 +/- 0.10 ab
Russet Norkotah 3 1.12 +/- 0.11 ab
Light Diploid (CH72.03) 3 0.91+/- 0.18 b

* Means followed by the same letter are not significantly different according
to Tukey's
5 hsd test at = 0.05.

TABLE 6: Length and similarity of plant class I c4h coding sequences to potato
Species Length Sequence Genbank
(bp) Identity bp Acc. #
(%)
Red Pepper (Capsicum annuum) 1518 1379 (91) AF212318
Lithospermum (Lithospermum erythrorhizon), c4h-2 1518 1249 (82) AB055508
Poplar (Populus x generosa) 1518 1200 (79) AF302495
Japanese Aspen (Populus kitakamiensis) 1518 1197 (79) D82815
Quaking Aspen (Populus tremuloides) 1518 1194 (79) U47293
Lithospermum (Lithospermum erythrorhizon), c4h-1 1518 1182 (78) AB055507
Grapefruit (Citrus x paradisi) 1518 1159 (76) AF378333
Chickpea (Cicer arietinum) 1518 1155 (76) AJO07449
Tree Cotton (Gossypium arboreum) 1518 1144 (75) AF286648
Madagascar Periwinkle (Catharanthus roseus) 1518 1143 (75) Z32563
Sweet Orange (Citrus sinensis), c4h-2 (Class 1) 1560 1113 (73) AF255014
Alfalfa (Medicago sativa) 1521 1112 (73) L11046
Zinnia (Zinnia elegans) 1518 1093 (72) U19922
Bishop's weed (Ammi majus) 1521 1088 (72) AY219918
Arabidopsis (Arabidopsis thaliana) 1518 1083 (71) U71080
Jerusalem Artichoke (Helianthus tuberosus) 1518 1073 (71) Z17369
Wild Licorice (Glycyrrhiza echinata) 1518 1018 (67) D87520

20


CA 02510950 2005-06-14

76
TABLE 7: Length and similarity of plant C4H amino acid sequences to potato
Species Length Sequence Identity Genbank
(amino acid) amino acid (%) Accession #
Red Pepper 505 441 (87) AAG43824
Lithospermum 505 425 (84) BAB71716
Madagascar Periwinkle 505 422 (83) CAA83552
Tree Cotton 505 420 (83) AAG10197
Wild Licorice 505 418 (82) BAA13414
Poplar 505 418 (82) AAG50231
Japanese Aspen 505 418 (82) BAA11579
Quaking Aspen 505 415 (82) AAB67874
Grapefruit 505 413 (81) AAK57011
Zinnia 505 407 (81) AAB42024
Arabidopsis 505 407 (81) AAB58355
Jerusalem Artichoke 505 407 (81) CAA78982
Chick Pea 505 406(80) CAA07519
Alfalfa 506 406 (80) S36878
Bishop's Weed 506 403 (80) AA062904


CA 02510950 2006-09-14

77
TABLE 8: The identification of conserved peptide domains by the alignment of
amino
acid sequences most highly homologous to potato C4H (SEQ ID NOS: 2, 29-34)
BOX A BOX B__
Potato M LLLEKTLIGLFFAILIAIIV KLRSKRFKL PGPIPVP FGNWLQVGD 51
Red Pepper M LLLEKTLVGLFFAIVVAIIV KLRSKRFKL PGPIPVP FGNWLQVGD
Lithospermum M LLLEKALIGLFFSFIIAIVI KLRGKKFKL PGPIPVP FGNWLQVGD
M. Periwinkle M LLLEKTLLGLFAAIIVASIV KLRGKKFKL PGPIPVP FGNWLQVGD
Tree Cotton M LFLEKVLISLFFTIIFAILV KLRGKRFKL PGPLPIP FGNWLQVGD
Wild Licorice M LLLEKTLLGLFIAAITAIAI KLRGRRFKL PGPIPVP FGNWLQVGD
Poplar M LLLEKTLLGSFVAILVAILV KLRGKRFKL PGPIPVP FGNWLQVGD
Potato DLNHRNLTEYAKKFGDVFLLRMGQRNLVVVSSPELAKEVLHTQGVEFGSRT 102
Red Pepper DLNHRNLTDYAKKFGDIFLLRMGQRNLVVVSSPESAKEVLHTQGVEFGSRT
Lithospermum DLNHRNLTEYAKKFGEIFLLRMGQRNLVVVSSPDLAKEVLHTQGVEFGSRT
M. Periwinkle DLNHRNLSDYAKKFGEIFLLRMGQRNLVVVSSPELAKEVLHTQGVEFGSRT
Tree Cotton DLNHRNLTDLAKKFGDIFLLRMGQRNLV'ISSPELAKEVLHTQGVEFGSRT!
Wild Licorice DLNHRNLTDLAKRFGIIIFLLRMGQRNLVVVSSPELAKEVLHTQGVEFGSRT
Poplar DLNHRNL'TDLAKKFGDIFLLRMGQRHLV`iVSSPDLSKEVLHTQGVEFGSRT
Potato RNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRGGWESE 153
Red Pepper RNWFDIFTGKGQD14VFTVYGEHWRKMnIMTVPFFTNKVVQQYRGGWESIN
Lithospermum RNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRKGWESE
M. Periwinkle RNVVFDIFTGKGQDMVFTVYGERWRKMRRIM2'VPFFFNKVVQQYRYGWEEE
Tree Cotton RNVVFDIFTGKGQDMVF'TVYGERWR1Q4RRIMTVPFE"KVVQQYRHGWEDE
Wild Licorice RNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRFGWESE
Poplar RNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYFIYGWEEE
Potato AASVVEDVKKNPESATNGIVLRKRLQLMMYNNMFRIMFDRRFESEDDPLFV 204
Red Pepper VASVVEDVKKNPESATNGIVLRKRLQLMMYNNMFRIMFDRRFESEDDPPFV
Lithospermum VESVIEDVKKIPESETVGIVLRKRLQLMMYNNMFRIMFDRRFESENDPLFM
M. Periwinkle AARVVEDVKKNPESATNGIVLRRRLQLMMYNNHYRIMFDRRFESEDDPLFV
Tree Cotton AASVVEDVKKNPEAATNGIVLRRKLQLMMYNNNYRIMFDRRFESEDDPLFV
Wild Licorice AASVVDDVRRNPDAAAGGIVLRRRLQLM4YNNMYRIMFDRRFESEEDPLFV
Poplar AAQVVEDVKKNPEAATNGIVLRRRLQLMMYNNMYRIMFDRRFESEDDPLFN
Potato KLRALNGERSRLAQSFEYNYGDFIPILRPFLRGYLKICKEVKEKRLKLFKD 255
Red Pepper KLRALNAERSRLAQSFEYNYGDFIPILRPFLRGYLKICKEVKEKRLQLFKD
Lithospermum KLRALNGERSRLAQSFDYNYGDFIPILRPFLRGYLKICKEVKETRLKLFKD
M. Periwinkle KLKALNGERSRLAQGFEYNYGDFIPILRPFLRGYLRICKEVKERRLQLFKD
Tree Cotton KLKALNGERSRLAQSFEYNYGDFIPILRPFLRGYLKLCKEVKEIRLQLFRD
Wild Licorice KLKALNGERSRLAQSFEYNYGDFIPILRPFLKGYLKICKEVKERRLKLFKD
Poplar KLKALNGERSRLAQSFDYNYGDFIPILRPFLRGYLKICQEVKERRLQLFKD
BOX
Potato YFVDERKKLANTKSMDSNALKCAIDHILEAQQKGEINEDNVLYIVENINV 306
Red Pepper YFVDERKKLSNTKSMDSNALKCAIDHILEAQQKGKINEDNVLYIVENINVV
Lithospermum YFVEERKKIASTKSTTTNGLKCAIDHILEAQQKGEINEDNVLYIVENINV
M. Periwinkle YFVDERKKFGSTKSMDNNSLKCAIDHILEAQQKGEINEDNVLYIVENINV
Tree Cotton QFLEERKKLATTKRIDNNALKCAIDHILDAQRKGEINEDNVLYIVENIN
Wild Licorice YFVDERMKLESTKSTSNEGLKCAIDHILDAQKKGEINEDNVLYIVENINV
Poplar YFVDERKKLASTKNMSNEGLKCAIDHILDAQKKGEINEDNVLYIVENINV


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TABLE 8:CONT.

C
Potato AIET LWSIEWGIAELVNHPHIQKKLRDEIDTVLGPGMQVTEPDMPKLPYL 357
Red Pepper AIET LWSIEWGIAELVNHPHIQQKLREEIDAVLGPGVQVTEPDTHKLPDL
Lithospermum AIET LWSIEWGIAELVNHPEIQKKLRDEIDTILGPGVQVTEPDTHKLPYL
M. Periwinkle AIET LWSIEWGIAELVNHPEIQKKLRDELETVLGPGVQITEPDTYKLPYL
Tree Cotton AIET LWSIEWGIAELVNHPEIQQKLRNEIDTVLGPGVQVTEPDTHKLPYL
Wild Licorice AIET LWSIEWGIAELVNHPEIQKKVRDEIDRVLGPGHQVTEPDMQKLPYL
Poplar AIET LWSIEWGIAELVNHPEIQKKLRHELDTLLGPGHQITEPDTYKLPYL
Potato QAVIKETLRLRMAIPLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNPA 408
Red Pepper QAVIKETLRLRMATPLLVPHMNIHDAKLAGYDIPAESKILVNPWWLANNPA
Lithospermum QAVIKETLRLRMAIPLLVPHMNLHDAKLNGYDIPAESKILVNAWWLANNPA
M. Periwinkle QAVIKETLRLRMAIPLFLPHMNLHDAKLGGYDIPAESKILVNAWFLANNPE
Tree Cotton QAVIKETLRLRMAIPLLVPHMNLHDAKLGGYDIPAESKILVNAWWLANNPA
Wild Licorice QAVIKETLRLRMAIPLLVPHMNLHDAKLGGYDIPAESKILVNAWWLANNPA
Poplar NAVIKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANNPA
BOX D
Potato HWKKPEEFRPERFFEEEKHVEANGNDFRFL FGVGRRSCPG ILALPILGI 459
Red Pepper HWKKPEEFRPERFLKEEKHVDANGNDFRFL FGVGRRSCPG ILALPILGI
Lithospermum QWKNPEEFRPERFLEEEAKVEANGNDFRYL FGVGRRSCPG ILALPILGI
M. Periwinkle HWKKPEEFRPERFLEEESKVEANGNDFRYL FGVGRRSCPG ILALPILGI
Tree Cotton HWKNPEEFRPERFFEEESKVEANGNDFRYL FGVGRRSCPG ILALPILGI
Wild Licorice NWKRPEEFRPERFLEEESHVEANGNDFRYL FGVGRRSCPG ILALPILGI
Poplar HWKNPEEFRPERFLEEEAKVEANGNDFRYL FGVGRRSCPG ILALPILGI
Potato TLGRLVQNFEMLPPPGQSKLDTSEKGGQFSLHILKHSTIVMKPRSF* 505
Red Pepper TLGRLVQNFELLPPPGQSKLDTTEKGGQFSLHILKHSTIVMKPRSF*
Lithospermum TLGRLVQNFELLPPPGQSKLDTSEKGGQFSLHILKHSTIVMKPRSF*
M. Periwinkle TIGRLVQNFELLPPPGKSKIDTSEKGGQFSLHILKHSTIVLKPRTF*
Tree Cotton TLGRLVQNFELLPPKGQSKLDTSEKGGQFSLHILKHSTIVAKPRVF*
Wild Licorice TLGRLVQNFELLPPPGQSKLDTAEKGGQFSLHILKHSTIVAKPRSF*
Poplar TLGRLVQNFELLPPPGQSKIDTAEKGGQFSLHILKHSTIVAKPRSF*



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TABLE 9: The volumes of 18s rRNA primer and competimer to obtain the
required 18s rRNA to competimer ratio.

Ratio 1:9 2:8 3:7
18s rRNA primer 1 p1 2 pl 3 yl
Competimer 9 p1 8 pl 7 pl

TABLE 10: Summary of statistical analysis on clones of diploid families; 13610-

T and 13395-B (Wang-Pruski, unpublished).
13610-T 13395-B
Mean 108.03 112.98
Standard Error 0.93 0.94
Standard Deviation 11.17 7.84
Dark
Kurtosis -0.42 -0.71
Skewness -0.09 0.10
Minimum 82.07 98.52
Maximum 134.48 132.41
Count 145 69
Confidence Level (95.0%) 1.83 1.88

25


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TABLE 11: ACD evaluation data for the selected diploid and tetraploid
samples.*

Jan 2003 Jan 2004 Feb 2004
Family Clone Number Degree Pixel pixel pixel
of ACD
density density density
13610 - T - 224 Dark 85.4 86.3 86.3
13610 - T - 154 Dark 89.3 82.8 81.9
13610 - T - 151 Dark 95.8 84.9 89.0
13610-T
13610 - T - 070 Light 140.1 128.6 136.0
13610 - T - 167 Light 137.3 132.6 125.4
13610 - T - 231 Light 133.0 137.8 133.2
13395 - B - 055 Dark 103.1 113.3 120.5
13395 - B - 052 Dark 105.2 100.1 111.3
13395 - B
13395 - B - 113 Light 123.3 127.7 124.1
13395 - B - 096 Light 122.0 132.1 111.4
Tetraploid Shepody Dark 131.2 132.2 120.6
Russet Burbank Light 124.2 126.9 129.7

5 *ACD light tubers are determined by higher pixel density values whereas ACD
dark
tubers have lower pixel density values.

15


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TABLE 12: Candidate genes and their full length cDNA sources used in this
study.
Name of gene candidates Plant Gene Sequences
Cinnamic acid 4-hydroxylase (C4H) Potato Identified at NSAC

TABLE 13: Designed primer set for differential expression analysis of C4H
gene.
Selected primer sets Tm C Length (nt)
Forward 5' - GAAGGGAGAGATCAACGAGG - 3'
60 20
Primer (SEQ ID NO: 27)
Reverse 5' - TTCTGCACCAAACGTCC -3'
57 17
Primer (SEQ ID NO: 28)

15
25


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TABLE 14. Mean ACD values of selected dark and light samples in family
13610-T grown and harvested in year 2002 and 2003
Mean ACD Mean ACD Mean ACD
value in Jan value in Jan value in Feb
13610-T
2003* 2004* 2004*
(MRD) (MRD) (MRD)
Dark 90.17 a 84.67 a 85.73 a
Light 136.80 b 133.00 b 131.53 b

Mean ACD Mean ACD Mean ACD
value in Jan value in Jan value in Feb
13395-B
2003* 2004* 2004*
(MRD) (MRD) (MRD)
Dark 104.15 a 106.7 a 115.90 a
Light 122.65 b 129.9 b 117.75 a
Mean ACD Mean ACD Mean ACD
value in Jan value in Jan value in Feb
Tetraploid
2003* 2004* 2004*
(MRD) (MRD) (MRD)
Shepody 131.20 a 132.20 a 120.60 b
Russet
Burbank 124.20 b 126.90 b 129.70 a
* Significant difference determined using one-way ANOVA at P<0.05 and
significances are shown as "a" and "b"



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TABLE 15: One-way ANOVA results of CgA, CA and CgA to CA ratio contents
in the dark and light clones of family 13610-T, 13395-B and tetraploid
samples.

Mean CgA Mean CA Mean CgA:CA
13610-T content content content
(mg 100"'g)* (mg 100"'g) * (mg 100"'g)*

Dark 0.49 a 808.15 a 6.06x 104 a
Light 0.24 b 833.47 a 2.88x10-4 b
Mean CgA Mean CA Mean CgA:CA
13395-B content content content
(mg 100"'g)* (mg 100''g) * (mg 100"'g)*
Dark 0.06 b 458.08 a 1.24x10' b
Light 0.47 a 393.33 a 11.95x10-4 a
Mean CgA Mean CA Mean CgA:CA

Tetraploid content content content
(mg 100"1g)* (mg 100'g) * (mg 100"'g)*
Shepody (Dark) 0.12 a 749.94 a 1.61x10-4 a
Russet Burbank
0.11 a 724.19 a 1.51 x 10-4 a
(Light)

* Small case "a" and "b" refers to significances from one-way ANOVA results at
P<0.05, the analyses were done for dark and light clones of each chemical
separately.


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84
TABLE 16: Normalized gene expression level of C4H in ACD dark and light
clones of 13610-T diploid family. (MPD - maximum pixel density)
PCR 1 PCR 2 PCR 3 PCR 4
Dark Light Dark Light Dark Light Dark Light
C41-1 (MPD) 120.35 23.46 83.08 10.97 79.35 29.06 108.68 8.90
Internal
25.35 25.64 19.56 11.48 34.33 39.16 77.48 62.15
Standard (MPD)
Blank (MPD 4.00 4.00 3.06 3.06 2.23 2.23 4.56 4.56
Normalized gene
expression 0.53 0.10 0.47 0.11 0.26 0.08 0.16 0.02
level*

Target gene MPD - Blank MPD
*Gene expression level = -----------------------------------------------------
X 1 : 9
Internal Standard MPD - Blank MPD

TABLE 17: Normalized gene expression level of C4H in ACD dark and light
clones of family 13395-B. (MPD - maximum pixel density)

PCR 1 PCR 2 PCR 3 PCR 4
Dark Light Dark Light Dark Light Dark Light
C4H (MPD) 58.57 22.97 44.97 32.92 45.83 22.41 61.76 28.72
Int. Std (MPD) 72.76 87.67 65.67 121.11 30 38.83 82.24 81.55
Blank (MPD 4.53 4.53 4.76 4.76 7.67 7.67 5.89 5.89
Normalized
gene expression 0.09 0.03 0.08 0.03 0.17 0.06 0.08 0.04
level*

Target gene MPD - Blank MPD

*Gene expression level = -----------------------------------------------------
X 1 : 9
Internal Standard MPD - Blank MPD



CA 02510950 2005-06-14

TABLE 18: Normalized gene expression level of C4H in Shepody and Russet
Burbank. (MPD - maximum pixel density)
5
PCR 1 PCR 2 PCR 3 PCR 4
Dark Light Dark Light Dark Light Dark Light
C41-1 (MPD) 210.41 89.25 190.4 142.62 180 32.11 129.14 56.91
Internal
57.45 88.41 16.03 35.63 70.62 45.11 85.92 192.47
Standard (MPD)
Blank (MPD 0.00 0.00 7.21 7.21 3.23 3.23 0.00 0.00
Normalized gene
0.41 0.11 1.32 0.44 0.28 0.08 0.17 0.03
expression level*

Target gene MPD - Blank MPD
*Gene expression level = -----------------------------------------------------
X 1 : 9
10 Internal Standard MPD - Blank MPD

20


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86
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15
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Forecasted Issue Date 2011-03-22
(22) Filed 2005-06-14
(41) Open to Public Inspection 2006-12-14
Examination Requested 2007-06-12
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Application Fee $400.00 2005-06-14
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Owners on Record

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Current Owners on Record
WANG-PRUSKI, GEFU
NARAYANAN, KARTHIKEYAN
BABIN, SANDRA ELIZABETH
Past Owners on Record
CANTLE, SANDRA
DALHOUSIE UNIVERSITY
HER MAJESTY THE QUEEN IN RIGHT OF THE PROVINCE OF NOVA SCOTIA, AS REPRESENTED BY THE NOVA SCOTIA AGRICULTURAL COLLEGE (NSAC)
NARAYANAN, KARTHIKEYAN
WANG-PRUSKI, GEFU
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