Note: Descriptions are shown in the official language in which they were submitted.
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GENE ISOLATED FROM RICINUS COMMUNIS ENCODING A NEW PRO-
TEIN THAT INTERACTS WITH THE OLEATE 12-HYDROXYLASE EN-
ZYME.
The present invention relates to the identifica-
tion and characterization of a gene of Ricinus communis
(R.communis) which encodes for a protein capable of in
teracting with the oleate 12-hydroxylase enzyme that
catalyzes the introduction of a hydroxyl group into the
molecule of oleic acid transforming it into ricinoleic
acid.
The invention also relates to means and methods
for producing transgenic plants with a modified compo-
sition of fatty acids.
Ricinoleic (12-hydroxy-9-octadecenoic) acid is a
monohydroxylated fatty acid whose only commercial
source is seed oil synthesized in the endosperm of ripe
seeds of R.communis, where it represents about 90% of
hydroxylated fatty acids.
Studies in vivo with radioactive tracers indicate
that, in the endosperm of unripe seeds of R.communis,
ricinoleic acid (also known with the term ricinoleate)
is synthesized by the direct substitution of a double
bond of oleic acid with a hydroxyl group (Morris, L.J.
1967, Biochem. Biophys. Res. Commun. 29, 311-315). This
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reaction is catalyzed by the oleate 12-hydroxylase en-
zyme whose activity seems to be associated with the en-
doplasmic reticulum.
Enzymatic tests indicate that the substrate for
oleate 12-hydroxylase is the oleic acid esterified with
lecithin or with another phospholipid; the esterified
ricinoleate is released from the lipid complex owing to
the intervention of a phospholipase A, specific for
fatty acids oxygenated in the presence of molecular
oxygen, NAD(P)H and cytochrome b5. NAD(P)H is required
for reducing the cytochrome b5. intermediate electron
donor for the hydroxylase reaction (Bafor M. Et al.,
(1991), Biochem J., 280, 507-514; Smith M.A. et al.,
(1992), Biochem J, 287, 141-144). The hydroxylated
fatty acid is then transferred, by means of the Kennedy
pathway, to the pathway of triacylglycerols where it
accumulates.
Ricinoleic acid, owing to the presence of the hy-
droxyl group, is one of the most versatile natural
products and has numerous industrial and food applica-
tions. In particular, ricinoleic acid can be used in
the production of paints, polymers such as nylon-11,
drugs, lubricants, cosmetics, resins and other materi-
als.
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The production of ricinoleic acid however is lim-
ited by the high susceptibility to climatic variations
of the cultivations of R.communis plants and by the
toxicity of ricin, an allergen present in castor beans.
The possibility of producing ricinoleic acid in
vegetable species which are more tolerant towards cli-
matic variations and which do not contain toxic sub-
stances would allow a larger and simpler production and
application of the acid itself.
For this purpose, the gene that encodes the oleate
12-hydroxylase enzyme has recently been isolated and
used to transform plants such as Nicotiana tabacum,
Arabidopsis thaliana, Linum usitatissimum and Brassica
napus.
In all these cases, however, although a modified
content of fatty acids was observed, the increase in
ricinoleic acid was low if not zero (Broun P. and
Somerville C., 1997, Plant Physiol, 113, 933-942).
It is known that in many biological processes,
such as replication, transcription or metabolism, enzy
matic complexes, whose action is correlated to the co
operation of various proteic subunits, intervene.
For example, evidence of the possible interaction
of various proteins in desaturation processes was ob-
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tamed from studies on the activity of the toluene 2-
mono-oxygenase enzyme isolated from the bacterium Burk-
holderia cepacia (Newman L.M, et al., 1995, Biochemis-
try, 34, 14066-14076).
It was therefore assumed that the hydroxylase of
vegetable fatty acids can also form part of a multicom
ponent system and that the hydroxylase activity of the
oleate 12-hydroxylase enzyme of R.communis consequently
requires the intervention of further co-factors or pro
teins.
A gene of R.communis which encodes for a new pro-
tein capable of interacting with the oleate 12-
hydroxylase enzyme, has now been identified and charac-
terized. This gene can be used in genetic transforma-
tion programs of plants containing the oleate 12-
hydroxylase enzyme to favour the production of ricino-
leic acid.
In accordance with this, an objective of the pres
ent invention is the cloned and sequenced gene which
encodes a protein capable of interacting with oleate
12-hydroxylase.
A second objective of the present invention is an
expression recombinant vector in host cells comprising
said gene.
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A further objective of the present invention is a
host microorganism transformed with said vector.
Yet another objective of the present invention re-
lates to transgenic plants transformed with said vec-
5 tor.
Additional objectives of the present invention
will appear evident upon reading the description and
examples.
Brief description of the figures
Figure 1: Southern Blot of the genomic DNA of different
species digested with the restriction enzyme EcoRI and
hybridized with the 762 by fragment of the plasmid
pTargl marked with 32P. The hybridization signal corre-
sponds to a gene in a single copy only evident in
R.communis.
Figure 2: Northern blot of messenger RNA extracted at
various development stages of the seed (10, 20, 30, 35
and 40 DAP), from the leaves, stem and roots of the
R.communis plant. The filter was hybridized with the
762 by fragment of the plasmid pTargl marked with 32P.
The presence of an mRNA with a molecular weight of
about 1.0 Kb, is observed in unripe seeds at 10 DAP and
20 DAP and a transcript with larger dimensions in the
leaves.
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Figure 3: Northern blot of messenger RNA extracted at
different development stages of the seed (10, 20, 30,
35 and 40 DAP) , from the leaves, stem and roots of the
R.communis plant. The filter was hybridized with the
1216 by fragment of oleate 12-hydroxylase marked with
s2P. The hybridization signal, of about 1.6 Kb, is pres-
ent in the unripe seeds at 20, 30, 35 and 40 DAP.
Detailed description of the invention
The isolation of nucleotide sequences which encode
for proteins of interest can be carried out by known
techniques.
In particular, to isolate new proteins that inter-
act with oleate 12-hydroxylase in unripe seeds of
R.communis, the "HybridZap two hybrid vector" of
Stratagene was used, a eukaryotic system (Saccharomy-
ces cerevisiae) which enables new genes to be identi-
fled in vivo, that encode proteins which interact with
a known protein (Fields S. et al., 1989, Nature, 340,
245-246). This system exploits the characteristics of
the transcriptional activator GAL4 of S.cerevisiae,
which regulates the expression of genes that encode en-
zymes involved in the galactose metabolism.
GAL4 consists of two domains separable and func-
tionally essential for its activity; an N-terminal do-
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main (Binding Domain, BD), which is linked to specific
sequences of the DNA (UAS: upstream activating se
quences), and a C-terminal domain, containing acid re
gions (Activation Domain, AD), which is necessary for
the transcriptional activation.
The system used allows two hybrid proteins to be
generated, containing the functional domains of GAL4,
i.e. the Binding Domain fused with a known protein
which acts as bait, and the Activation Domain fused
with unknown proteins (target) from an expression li-
brary.
If the known protein interacts with a target pro-
tein forming a protein-protein complex, the two func-
tional domains of GAL4 are brought under optimum condi-
tions and activate the transcription of the reporter
gene lac-Z, whose product is shown by means of colori-
metric reaction.
The total RNA was extracted from a pool of unripe
seeds of R.communis using the "Hot-Phenol" method and
the polyadenilate messenger RNA (mRNA) was isolated
with oligo-dT columns (Pharmacia). The cDNA encoding
the oleate 12-hydroxylase enzyme was subsequently pre-
pared by applying the polymerase chain reaction (PCR)
technique on the mRNA using, as primers, a pair of oli-
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gonucleotides having the sequences that flank the en-
coding region comprising the start and stop translation
codon of said gene.
As the interaction of target proteins can take
place with the whole protein under examination or with
parts of it, it was decided to clone both the whole
gene encoding for oleate 12-hydroxylase and its termi
nal regions 5' and 3', in the plasmid pBD-GAL4, which
contains the sequence that encodes for the Binding Do
main.
The three DNA inserts were first amplified with
the appropriate primers having restriction sites EcoRI
for the Forward primer and SalI for the Reverse primer,
digested with the above enzymes, directionally inserted
into the vector pBD-GALA4 predigested with the same en-
zymes and introduced into the competent cells Epicurean
coli XL1-Blue.
The recombinant clones, containing the expected
fragments, were characterized by means of restriction
analysis and their identity was confirmed with the se
quence reactions carried out using the Taq Dye Deoxy
Terminator Cycle Sequencing kit (Perkin Eimer) and ana-
lyzed with the automatic sequencer. A cDNA library was
then prepared from unripe seeds of R.communis in the
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HybridZap phage vector which expresses consistent hy-
brid proteins of the activation domain of GALA4 and
proteins of R. communis .
In practice, the polyadenilate mRNA of R.communis
was used for the synthesis of double filament cDNA op
erating according to the protocols suggested by the Kit
distributor (Stratagene).
The molecules of cDNA having a high molecular
weight, useful for the construction of the library,
were then separated from those having a low molecular
weight, which represent the fraction of molecules in
which the synthesis is incomplete.
The fraction of high molecular weight cDNA was in-
serted into the ~. HybridZap phage vector and packed
with the packaging extracts containing proteins for the
head and tail of the phage. The dimensions of the in-
serts present in the library produced were checked by
means of PCR and amplified with a specific pair of
primers for the vector pAD-GAL4. The results obtained
demonstrated that the fragments of the cDNA library had
an average dimension of 1.4 Kb.
After amplification, the library in the lambda
phage was converted to a plasmid library by excision in
vivo.
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The library was subsequently multiplied in the
strain of E.coli XLOR and the plasmid DNA was extracted
and co-transformed, in separate co-transformation proc-
esses, with the DNA extracted from the bait plasmids
5 containing the whole gene and parts of oleate 12-
hydroxylase, using yeast cells (YRG-2 strain), having
reporter genes his3 and lacZ.
From the test of the three sequences of oleate 12
hydroxylase with the expression library, colonies of
10 yeast were identified which had the typical blue colour
of the lacZ gene activity, thus demonstrating the prob-
able interaction of the bait protein with an unknown
target protein.
Subsequent analyses on these colonies enabled a
"positive" co-transformed yeast clone to be identified,
which activated the transcription of both reporter
genes. This indicated the complete interaction between
the N-terminal region of oleate 12-hydroxylase and an
unknown Target protein.
The plasmid isolated from this positive yeast
clone was indicated with the abbreviation pTargl.
The interaction specificity between oleate 12-
hydroxylase and the new protein identified, was con-
firmed by co-transformation experiments in S.cerevisiae
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YRG-2 yeast cells with bait plasmids containing the
whole oleate 12-hydroxylase gene and the 5' terminal
portion of this gene, respectively. Two yeast colonies
were identified from tests of the two sequences, one
for each co-transformation process, which had the typi-
cal blue colour of the lacZ gene activity.
To confirm the presence and dimensions of the bait
proteins and to verify the presence and dimensions of
the gene encoding the protein Targl2 identified, PCR
analyses were carried out on the DNA extracted from the
yeast clones resulting positive from the expression
test of the reporter gene lacZ, using specific primers
for the Binding Domain region and for the Activation
Domain region.
The authenticity of the amplification products ob-
tamed was demonstrated not only by the size of the ex-
pected fragments, but also by the hybridization analy-
sis carried out on the latter.
In addition, the plasmid DNA of pTargl was iso
lated from the yeast colony and the cDNA insert was pu
rified with the "Double GeneClean" kit (BIO 101 Inc.,
U.S.A.). The purified fragment was subsequently cloned
in the vector pGEM-T (Promega) and then introduced into
competent cells of E.coli DHSa.
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The plasmid DNA isolated from the recombinant
clones was subjected to sequence analysis with a Taq
Dye Deoxy Terminator Cycle Sequencing Kit (Perkin
Elmer), using the automatic sequencer ABI 373A (Perkin
Elmer).
From the sequence analyses carried out on the DNA
of the insert of the plasmid pTargl, it can be observed
that the fragment isolated has a dimension of 762 by
and contains an Open Reading Frame (ORF) of 540 by pre-
ceded by 75 by at 5' and followed by 147 by at 3'. The
ORF encodes a protein of 180 aminoacids with a molecu-
lar weight of 19.8 KDa.
The nucleotide and aminoacid sequences were com
pared with the sequences available on data banks by
means of FASTA and BLAST analyses, and homologous se
quences were not found, indicating the uniqueness of
the DNA tract of R.communis and the uniqueness of the
protein identified.
To verify the identity of the protein capable of
interacting with oleate 12-hydroxylase, analyses were
carried out on the genomic DNA of different species.
The results showed the presence of a signal only in
R.communis. This demonstrates that the gene isolated is
specific of the genome of R.communis and is not an out
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come of the system adopted for its identification.
In addition, expression analyses were carried out
on the messenger RNA extracted at different development
stages of the seed (10, 20, 30, 35 and 40 days after
pollination), from the leaves, stem and roots of the
R. communis plant .
The results of the hybridization of the Northern
blot with the fragment of pTargl marked with 32P showed
the gene expression in the leaves and unripe seeds 10
and 20 days after pollination.
The same filter was hybridized with the fragment
of oleate 12-hydroxylase which begins to be expressed
in the unripe seeds at 20 DAP, where the signal is very
weak, subsequently increasing its expression in the
stages at 30, 35 and 40 DAP. As expected, there was no
hybridization signal in the samples of RNA correspond-
ing to leaves, stem and roots.
On the basis of these results, it can be concluded
that the new protein most probably intervenes in the
first development stages of the R.communis seed at the
beginning of the synthesis of ricinoleic acid, carrying
out a regulating action. The gene of the present inven-
tion can be cloned in an expression vector in plants,
by putting it under the control of suitable regulation
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sequences (promoter and terminator).
Vectors suitable for the purposes of the present
invention are for example those deriving from the Ti
plasmid of Agrobacterium tumefaciens as described by
Bevan M., (1984), Nucleic Acid Research 12: 8711-8721.
These vectors are used to transform the plants by
means of conventional methods. The method described by
G. An et al. (Binary vectors, Plant Molecular Biology
Manual A3, Kluwer Academic, Dordrecht, pages 1-19,
1988), which is based on the capacity of Agrobacterium
tumefaciens to transfer part of its own DNA to vegeta-
ble cells, is preferably used.
The plasmid pTargl containing the gene of the pre-
sent invention was deposited as E.coli DHSa/MA292 at
the CentraalBureau Voor Schimmelcultures where it re
ceived the deposit number CBS 101642.
The following examples, whose sole purpose is to
provide a more detailed description of the present in
vention, should in no way be considered as limiting the
scope of the invention itself.
Example 1
RNA Isolation
The total RNA was extracted from a pool of unripe
seeds (10-40 days after pollination) of Ricinus commu-
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nis by means of the "Hot-Phenol" method described by
Shirzadegan M. et al. (1991), Nucl. Acids Res.: 19,
6055, to which several modifications were made.
In short, 2 g of vegetable material were crushed
5 in liquid nitrogen and then suspended in 6 ml of ex
traction buffer (0.1 M LiCl, 0.1 M Tris-HC1 pH 7.6,
0.01 M EDTA, to Sodium dodecylsulfate (SDS), phenol)
preheated to 80°C. The suspension was incubated at 80°C
for 5 minutes and 3 ml of a mixture of chloro
10 form/isoamyl (24:1, v/v) were added. The sample was
vortex mixed and subsequently centrifuged at 12,000
rpm, at 4°C for 15 minutes. The aqueous phase was re-
covered, subjected to an additional extraction cycle
with phenol/chloroform/isoamyl (25:24:1) and centri-
15 fuged under the same conditions specified above. The
supernatant was recovered and the RNA precipitated by
the addition of a volume of LiCl 4 M. The sample was
incubated at -20°C for a night and then centrifuged at
13,000 rpm, at 4°C for 30 minutes.
The RNA pellet was re-suspended in water, trans-
ferred to microcentrifuge tubes and precipitated by the
addition of LiCl 4 M and 0.2 volumes of EDTA 0.5 M. Af-
ter centrifugation at 13,000 rpm, at 4°C for 30 min-
utes, the pellet was suspended again in water and pre-
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cipitated with 0.1 volumes of NaCl 5 M and 2.5 volumes
of ethanol 1000. After centrifugation at 15,000 rpm, at
4°C for 30 minutes, the pellet was recovered, washed
twice with ethanol 700, dried and re-suspended in wa-
ter.
Example 2
Isolation of the cDNA encoding for oleate 12-
hydroxylase
The polyadenilate messenger RNA (poliA-RNA) was
prepared from the total RNA obtained in example 1, us
ing oligo-dT columns (Pharmacia) according to the in
structions of the distributor.
3.5 ~g of polyadenilate messenger RNA were then
used for the synthesis of double filament cDNA using
the Kit distributed by PHARMACIA, operating under the
experimental conditions suggested by the supplier of
the kit.
On the basis of the sequence of oleate 12-
hydroxylase deposited in the data bank (GenBank, AC
U22378) the following oligonucleotides were synthe-
sized:
(1) 5'GGA TCC CTC AGG AAA GTG CTT A 3' (FORWARD)
(2) 5' TCT AGA CAT TCC TTC TTG TTC TAA TT3' (REVERSE)
These oligonucleotides, which correspond to the
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regions that flank the portion encoding the enzyme com
prising the start and stop translation codon, were used
as primers for the isolation of the fragment corre
sponding to oleate 12-hydroxylase by means of the poly
merase chain reaction (PCR) technique.
The amplification was effected in a DNA Thermal
Cycler 480 apparatus (Perkin Elmer Cetus) using a reac-
tion mixture (25 ~1) containing 6 ~1 of double filament
cDNA, 10 mM Tris HC1 pH 8.3, 1.5 mM MgClz, 50 mM KCl,
2 . 5 ~M of each primer, 0 . 1 mM of dNTP and ?_ . 5 polymer-
ase Taq Units (Boheringer).
After a first denaturation cycle for 5 minutes at
95°C, the reaction was continued with the following cy-
cles:
1 minute at 94°C (denaturation)
1 minute at 56°C (pairing)
2 minutes at 72°C (lengthening)
for a total of 35 cycles, followed by 10 minutes at
72°C (final extension).
The amplification product, corresponding to a
fragment of about 1200 base couples, was separated on
agarose gel at 1.0o, the DNA band of interest was re-
covered and purified with the GeneCleanTM kit (BIO 101
Inc, U.S.A.). About 100 ng of the DNA thus isolated
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were ligated to 50 ng of pGEM-T plasmid (Promega) in 10
~1 of reaction mixture, in the presence of 2 units of
T4 DNA ligase, at 4°C for a night.
~1 of this mixture were used to transform compe-
5 tent cells of E.coli DH5a (BRL). The transforming
agents were selected on plates of LB medium (NaCl 10
g/1, Yeast extract 5 g/1, Bacto-triptone 10 g/1 and
agar 20 g/1) containing 50 ~tg/ml of ampicillin.
The plasmid DNA extracted from 6 positive clones
was subjected to sequence analysis to verify the nu-
cleotidic correspondence with the gene of oleate 12-
hydroxylase isolated by Van de Loo F. Et al., 1995,
PNAS, 92, 6743-6747. The reactions and sequence analy-
ses were carried out with the Taq Dye Deoxy Terminator
Cycle SequencingTM kit (AB-PEC) using an ABI Prism 373A
DNA Sequencer (AB-PEC).
One of the plasmids analyzed, containing a frag-
ment of DNA analogous to the published sequence SEQ:ID
Nr. l, was called pCl8-MA.
Example 3
Construction of the "bait" vectors (pBD-GALS
As the interaction of target proteins can take
place with the whole protein under examination (bait)
or parts of this, it was decided to clone both the
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fragment corresponding to the whole gene encoding for
oleate 12-hydroxylase, and also the fragments corre-
sponding to the regions 5' and 3' of said gene, in the
plasmid pBD-GAL4 (Stratagene).
For this purpose four oligonucleotides were syn-
thesized, of which the Forward primers (abbreviated as
F) have the restriction site EcoRI and the Reverse
primers (abbreviated as R) the site SalI. The nucleo-
tide sequences of the primers are as follows:
(a) 5'GAA TTC CGC ATG TCT ACT GTC 3' (Forward, HydGal-
F)
(b) 5'GTC GAC CAT TCC TTC TTG TTC 3' (Reverse, HydGal-
R)
(c) 5'GTC GAC GCG ATC GTA AGG 3' (GalHydi-R) and
(d) 5'GAA TTC AAT GTC TCT GGT AGA C 3' (GalHydi-F)
The primers HydGal-F and HydGal-R were used to am-
plify the whole gene of oleate 12-hydroxylase.
The primers HydGal-F/GalHydi-R and HydGal
R/GalHydi-F were used to amplify the region 5' of 624
by (SEQ: ID Nr:2) and the region 3' of 633 by (SEQ: ID
Nr:3) of the gene of oleate 12-hydroxylase, respec-
tively.
The amplifications with the above primers were
carried out on 20 ng of the fragment of oleate 12-
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hydroxylase previously cloned and sequenced.
The amplification products having the expected di-
mensions were digested with 10 units of restriction en-
zymes EcoRI and SalI (Boheringher), separated on aga-
5 rose gel to and the fragments of DNA of interest were
then recovered and purified with the GeneCleanTM kit
(BIO 101 Inc.).
About 100 ng of each fragment were ligated, sepa
rately, with the plasmid pBD-GAL4 linearized with the
10 enzymes EcoRI and SalI, in 10 ml of reaction mixture,
in the presence of 2 units of T4 DNA ligase, at 4°C for
a night. The ligase mixtures were used to transform
competent cells of Epicurian coli XL1 Blue (Strata-
gene). The recombinant clones were selected on plates
15 of LB medium to which 30 ~g/ml of chloramphenicol had
been added. The following recombinant "bait" plasmids
were identified, which contain a fragment which con-
sists of the Binding Domain of Gal4 condensed with:
(a) the whole gene of oleate 12-hydroxylase (pBD-
20 GALA4/C18)~
(b) the 5-terminal region of the gene of oleate 12-
hydroxylase (pBD-GALA4/C18-5); and
(c) the 3-terminal region of the gene of oleate 12-
hydroxylase (pBD-GALA4/C18-3).
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The plasmids were characterized by restriction
analysis and their identity was confirmed by effecting
sequence reactions with the Taq Dye Deoxy Terminator
Cycle sequencing kit (Perkin Elmer) and analyzed with
the automatic sequencer ABI 373A (Perkin Elmer).
Example 4
Construction of the target librar
For the construction of a "target" library made up
of hybrid proteins consisting of the activation domain
of GALA4 and proteins of unripe seeds of R.communis,
the HybridZap phage vector was used. The experimental
conditions adopted were those suggested by the supplier
of the kit (Stratagene, HybridZapTM Two-Hybrid cDNA
gapack cloning kit, catalogue Nr.: 235612).
About 5 ~g of polyadenilate mRNA of R.communis
were used for the synthesis of the double filament
cDNA.
The ends of the cDNA molecules, to which 3.6 ~.g of
a linker having the restriction site EcoRI had been
added,
were then blunted by the action of the polymerase DNA
Pfu (Stratagene), and subjected to digestion with the
enzyme XhoI (120 units), whose site is present in the
primer polydT used for the synthesis of the first cDNA
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filament. This gave rise to molecules having the EcoRI
site at one end and the XhoI site at the other end.
The sample of cDNA was then passed on a Sephacryl
S-500 column equilibrated in 20 mM Tris-HC1 pH 7.5, 10
mM EDTA, 100 mM NaCl, and subjected to centrifugation
for 2 minutes at 400 revs.
Three fractions were recovered, whose molecular
weight was verified by means of electrophoresis on non-
denaturing polyacrylamide gel at 50 (Sambrook, J. Et.
A1., (1989), Cold Spring Harbor Laboratory Press).
About 100 ng of the first fraction of cDNA, corre-
sponding to that with a high molecular weight, were 1i-
gated with 1 ~g of the ~ HybridZap phage vector predi-
Bested with the enzymes EcoRI and XhoI and packed with
packaging extracts containing proteins for the head and
tail of the phage. The total quantity of phage parti-
cles obtained by the packaging in vitro was determined
by plating aliquots with the strain XL1-Blue MRF'.
The primary library obtained contains a total of
1.4 x 106 plaque forming units (pfu) per ~tg of ligated
vector arms and 97~ of these contains the DNA insert.
The dimensions of the library produced were verified by
subjecting to PCR reaction, 20 phage plaques, selected
at random, and amplified with the specific pair of
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primers for the vector pAD-GAL4:
a) 5'AD primer: 5' AGG GAT GTT TAA TAC CAC TAC3'
b) 3'AD primer: 5' GCA CAG TTG AAG TGA ACT TGC3'
The results obtained showed that the inserts of the
cDNA library had an average dimension of 1.4 kb.
Example 5
Conversion of the HybridZap phagic library to the pAD-
GALA4 plasmid library
The primary library constructed in the ~. HybridZap
phage was converted to the pAD-GAL4 plasmid library by
means of total excision in vivo according to the method
described by the supplier of the kit adopted (Strata-
gene ) .
For the amplification of the primary library, 1x106
phages were used to infect the host cells XL1 Blue
MRF', which, by enabling replication inside the phages,
after their lysis, allowed a library consisting of
about 1x109 phagic particles to be recovered.
An aliquot of the amplified library (1x108 pfu) was
then incubated with 1x101° pfu of ExAssist helper phage
and 1x109 of XL1 Blue MRF' cells to generate particles
of phagemid containing the plasmid vector excised from
the phage vector.
The excess number of phage helpers and bacterial
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cells with respect to the number of phages of the li-
brary was used to ensure that each cell was infected
both by the phage helpers and by the lambda phage, thus
obtaining an effective and representative excision in
vivo.
The incubation of the lambda phages with the phage
helpers and XL1 Blue MRF' cells took place at 37 °C for
minutes, after which 20 ml of LB medium were added
and the incubation was continued at 37°C for a further
10 3 hours.
In order to lyse the phage particles and enable
the particles of phagemid to be released and recovered,
the suspension was incubated at 70°C for 20 minutes and
then centrifuged for 10 minutes at 500 revs. The super-
15 natant (phagemid stock) was recovered and conserved at
4°C.
Cells of E.coli XLOR were incubated with 1x10$ of
phagemid in a ratio of 10:1, at 37°C for 15 minutes.
500 ml of LB medium containing 50 ~g/ml of ampicillin
were then added and the incubation was continued at
37°C for 3 hours.
This operation allowed cells to be obtained, con-
taming the target library in a plasmid vector.
The suspension was centrifuged for 10 minutes at
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
500 revs and the plasmidic DNA was isolated from the
pellet according to the protocol of alkaline lysis sug-
Bested by Sambrook, J. Et al., 1989, Cold Spring Harbor
laboratory Press.
5 Example 6
Screening of the library
To identify the "target" proteins which interact
with the "bait" protein, the yeast strain S.cerevisiae
YRG-2, containing the reporter genes his3 and lacZ, was
10 co-transformed with the DNA prepared from the bait
plasmids and the DNA isolated from the target plasmid
library.
About 5 ~.g of DNA extracted from the plasmids pBD-
GALA4/C18, pBD-GALA4/C18-5' and pBD-GALA4/C18-3', were
15 co-transformed, in separate co-transformation proc-
esses, with 10 ~.g of DNA extracted from the target li-
brary. The co-transformations were plated on SD selec-
tive medium (yeast nitrogenated base without aminoacids
6.7 g/1, D-sorbitol 182.2 g/1, agar 20 g/1, 100 ml of
20 the appropriate aminoacidic solution concentrated lOx
and glucose 2%) without leucine aminoacids (Leu ), tryp-
tophan (Trp ) and histidine (His ), which allows only
the growth of the yeast colonies containing both of the
recombinant plasmids pBD-GALA4(Trp ) and pAD-GAL4 (Leu
CA 02374015 2001-11-15
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26
), and incubated at 30°C for 5 days. The yeast colonies
thus obtained were transferred to Watman 3MM filter pa-
per and subjected to the expression test for the re-
porter gene lacZ, by soaking the filters in a solution
containing the chromogenic substrate 5-bromo-4-chloro-
indolyl ~3-D-galactoside (X-Gal) and incubating at 30°C
for a night.
From the test of the 3 sequences of oleate 12-
hydroxylase with the library, 7 yeast colonies were
identified, having the typical blue colour of the ac-
tivity of the gene lacZ, thus indicating the probable
interaction of the bait protein with an unknown target
protein. To verify the authenticity of the above colo-
nies, these were re-plated on selective medium and the
X-Gal test was repeated.
Of the 7 colonies identified, only one reconfirmed
the previous phenotype, whereas the others proved to be
false positives.
The positive yeast colony proved to come from the
co-transformation process in which the plasmid pBD-
GAL4/C18-5' was used, containing the 5' region of the
oleate 12-hydroxylase gene.
Transformation control experiments were parallelly
carried out with the 4 plasmids pGal4, p53, pSV40 and
CA 02374015 2001-11-15
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27
pLaminC (Stratagene). These plasmids were used singly
or in pairs as indicated in table 1 and, on the basis
of the combinations, they acted as positive or negative
controls. Table 1 also indicates the results of the
transformation processes in which the bait protein in-
teracts with an unknown target protein.
Table 1
Transformations SD1 SD2 SD3 SD4
GROWTH
pGAL4 blue
p53 white
pSV40 white
pLaminC white
p53 and pSV40 blue blue
pLaminC and pSV40 white 0
pBDGAL4-Bait white
pBDGAL4-Target white
pBDGAL4-Target pBDGAL4-Bait white white blue blue
wherein SD1 - SD medium without Leu; SD2 - SD medium
without Trp; SD3 - SD medium without Leu and Trp and
SD4 - SD medium without Leu, Trp and HIs.
Example 7
Verification of the interaction specificit
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28
To verify the interaction specificity between ole-
ate 12-hydroxylase and the new protein identified, co-
transformation experiments were carried out in yeast in
which the DNA of the target plasmid, called pTargl, was
tested in combination with the whole gene of oleate 12-
hydroxylase, with the 5' region and with the genes of
the bait proteins p53 and pLaminC.
To isolate the plasmid DNA pTargl from the yeast
colony resulting positive from the X-GAL test, the lat-
ter was inoculated into 2 ml of YPAD growth medium
(peptone 20 g/1, yeast extract 10 g/1, adenine sulfate
40 mg/1 and glucose 20) and incubated at 30°C for 2
days.
To recover the plasmid DNA the cellular wall of
the yeast was mechanically broken using glass balls and
the nucleic acids were precipitated following the pro-
cedure described by Sambrook, J. Et al., 1989, Cold
Spring Harbor Laboratory Press.
About 5 ~g of the plasmid pTargl were used in a
co-transformation experiment with 5 ~g of DNA of the
plasmid containing the whole oleate 12-hydroxylase gene
(pBD-GAL4/C18), and in another experiment with 5 ~g of
DNA of the plasmid containing 5-terminal portion of the
oleate 12-hydroxylase gene (pBD-GAL4/C18-5'). 2 yeast
CA 02374015 2001-11-15
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29
colonies were identified from the tests of the two se
quences, one for each co-transformation process, which
had the typical blue colour of the activity of the lacZ
gene and thus indicating the interaction of the bait
protein with an unknown target protein.
The above colonies were plated on selective SD me-
dium (Leu , Trp , His ) and tested again with X-Gal. The
two colonies identified reconfirmed the previous pheno-
type.
When, on the other hand, the target protein was
tested with the bait proteins p53 and pLaminC, as ex
pected, none of the colonies had the blue colour with
the X-Gal test. These results indicated that the new
protein identified interacted specifically with oleate
12-hydroxylase.
To confirm the presence and dimensions of the pro-
teins used as bait and to verify the presence and di-
mensions of the fragment encoding for the target pro-
tein, called TargHl2, PCR analyses were effected on the
DNA extracted from the two yeast clones resulting posi-
tive from the expression test of the reporter gene
lacZ.
Specific primers for the Binding Domain (BD) re-
gion and for the Activation Domain (AD) region were
CA 02374015 2001-11-15
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used for the amplification under the conditions sug-
Bested by the kit supplier.
The sequences of the specific primers for the Ac-
tivation Domain were specified in example 4, whereas
5 the sequences for the Binding Domain are the following:
a) 5'BD: 5'GTG CGA CAT CAT CAT CGG AAG3'
b) 3'BD: 5'CCT AAG AGT CAC TTT AAA ATT3'
The authenticity of the amplification products was
demonstrated by the size of the expected fragments and
10 also by the hybridization analysis carried out on
these.
Example 8
Sequence analysis of the plasmid Targ1
The plasmid DNA of pTargl was subjected to ampli-
15 fication reaction with the use of specific primers of
the plasmid pAD-Gal4, 5'AD primer and 3'AD primer. The
fragment produced was purified with the GeneCleanTM Kit
(BIO 101 Inc. U.S.A.). About 100 ng of the DNA thus pu-
rified were ligated with 50 ng of the plasmid pGEM-T
20 (Promega) in 10 ~1 of reaction mixture, in the presence
of 2 units of T4 DNA ligase, at 4°C for a night.
5 ~g of the ligase mixture were used to transform
competent cells of E.coli DHSa (BRL). The transforming
agents were selected on LB medium to which 50 ~g/ml of
CA 02374015 2001-11-15
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31
ampicillin had been added.
The plasmid DNA was extracted from 6 positive
clones, i.e. showing a white colour, and sequenced with
the Taq Dye Deoxy T Cycle SequencingTM Kit (AB-PEC), us
ing an ABI Prism 373A DNA Sequencer (AB-PEC).
From the sequence analyses, it can be observed
that the isolated gene of 762 by (SEQ: ID Nr:4) con-
tains an Open reading frame (ORF) of 540 bp, preceded
by 75 by at 5' and followed by 147 by at 3' where the
poly (A) tail is present. The ORF encodes a protein of
180 aminoacids (SEQ: ID Nr:5), indicated as TargHl2,
with a molecular weight of 19.8 kilodaltons.
Example 9
Southern Blot analysis
To verify the identity of the protein capable of
interacting with oleate 12-hydroxylase and the number
of ricin copies of the gene corresponding to the insert
of the plasmid pTargl, analyses were carried out on the
genomic DNA of different species, isolated with the
method of Dellaporta et al., 1983, Plant Mol. Biol.: 1,
19-21.
About 6 ~g of genomic DNA isolated respectively
from:
Ricinus communis, Lesquerella fendleri, Linum usitatis-
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32
simum, Brassica napus, Helianthus annus, Limnantes
douglasii, Lycopersicon esculentum, Beta vulgaris, Zea
mays, Nicotiana tabacum and Saccharomyces cerevisiae,
were digested with 100 units of the enzyme EcoRI (Boe-
hringer) in 100 ~1 of reaction mixture, at 37°C for 1
hour.
The digestion mixtures were charged onto agarose
gel at 0.8o and subjected to horizontal electrophore-
sis. The DNA was transferred onto nitrocellulose filter
(Hybond-N(+)R, Hamersham) with the Southern method (Sam-
brook, J. Et al., Cold Spring Harbor Laboratory Press).
This filter was hybridized for a night at 65°C,
after a pre-hybridization of about 4 hours at 65°C, in
a solution containing 6XSSC, to SDS, lOX Denhardt's and
tRNA at a concentration of 10 ~g per ml of hybridiza-
tion solution used. The fragment of 762 by isolated
from the plasmid pTargl, marked with 3'P (Sambrook and
Fritsch, E.F. and Maniatis, T. (1989), Molecular Clon-
ing: A Laboratory Manual, (Cold Spring Harbor Lab.,
Cold Spring Harbor, NY) 2nd Ed. 1989), was used as
probe.
The filter was washed twice at 65°C in 2XSSC, 0.20
SDS for 30 minutes and once in 0.2XSSC, 0.020 SDS for
20 minutes, then exposed to X-rays by autoradiography.
CA 02374015 2001-11-15
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33
The hybridization showed a single band only on the
genomic DNA isolated from R.communis. This demonstrated
that the gene isolated was specific of the genome of
this plant, which was present in a single copy and that
the result obtained was not an outcome of the system
used for its identification (figure 1).
Example 10
Expression analysis
To effect the expression analysis of the gene en-
coding the protein TargHl2, the messenger RNA was pre-
pared from different organs (leaves, stem and roots) of
R.communis and at different development stages of the
seed (10-20-30-35-40 days after pollination, DAP).
About 7 ~g of each sample were charged onto aga-
rose gel at 1.4o containing formaldehyde and subjected
to an electrophoretic run. The RNA were transferred
onto a nitrocellulose filter (Hybond-N(+)R Hamersham)
using the standard Northern blot procedure. The filter
was hybridized with a probe corresponding to the frag-
ment of 762 by of the plasmid pTargl marked with 32P.
The reaction was carried out in a hybridization solu-
tion containing 6XSSC, loSDS, lOX Denhardt's and 10 ~.g
of tRNA, at 65°C for a night, after a pre-hybridization
of 5 hours in the same solution at 65°C.
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34
The filter was washed twice at 65°C in 2xSSC,
0.2oSDS for 20 minutes and once in 0.2XSSC, 0.02oSDS at
65°C for 20 minutes and then exposed to X-rays by auto-
radiography.
The results showed the presence of a signal, lo-
calized in a band of about 1 Kb (figure 2), in the sam-
ples corresponding to unripe seeds of 10 and 20 DAP. A
similar signal was observed in the sample of RNA ex-
tracted from the leaves.
The same Northern filter was hybridized with the
fragment of oleate 12-hydroxylase which begins to ex-
press itself in the unripe seeds at 20 DAP, where the
signal is very weak, subsequently increasing its ex-
pression in the stages at 30, 35 and 40 DAP (figure 3).
No hybridization signal was observed in the samples of
RNA corresponding to the leaves, stem and roots.
From these results it can be deduced that the pro-
tein isolated with the double hybrid system most proba-
bly intervenes in the first stages of development of
the seed of R.communis, at the beginning of the synthe-
sis of ricinoleic acid.
CA 02374015 2001-11-15
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EUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
RECEIPT IN THE CAS OF AN ORIGINAL DEPOSIT
Metapontum Agrobios
S.$.iOnICa 106 km. 448,2 I issued pursuant to Rule 7.1 by the
I 7~JO1O Mi_TAPONTO MT ~ INTER"IATIONAL DEPCSITARY AUTHORITY
identified at the bottom of this page
~ta~l~
name and address of depOSltor
I. IDENTIFICATION OF T~ M=CROORGANISM
Identification reference given by the ~ Accession number given by the
DEPOSITOR: ~ INTERNATIONAL DEPOSITARY AUTHORITY:
MA292 I CBS 101642
II. SCIENTIFIC DESG~PTION AND/OR PROPOSED TAXONOMIC DESIQ~1TION I
The microorganism identified under I above was accompanied by:
I I
I ~ a scientific description
I ~ a proposed taxonomic designation
(mark with a cross where applicable)
III. RECEIPT AND ACCEPTANCB
j This International Depositary
accepts the microorganism identified
under I above, which
I
received by it on ~3..~ (date
dd-mm-yy
of
the
original
deposit)
1
IV. RECEIPT OF REQUEST FOR CONVERSION
I The microorganism identified was
under I above received
by
this
International
Depositary
Authority on not applicable (datedd-mm-yy
of
the
original
deposit)
and
a
I
request to convert the original a
deposit to deposit
under
the
Budapest
Treaty
was
received
by it on . notappliCable (date dd-mm-yy
of
receipt
of
request
for
conversion)
I V. ?NTBRt~ITIONAL DEPOSITARY
11DTHORITY
Name : Centraalbureau voo~ Schimmelcuttures signature ( s ) of person ( s )
having the power
j to represent the International
Depositary
Authority authorized offi 'al
Address : Oosterstraat 1
P.O. Box 273
3740 AG BAARN Mrs F.B. Snippe-Claus JA S rs
The Netherlands
Date (dd-mm-yy): 1 3-99
I
1 Where Rule 6.9(d) applies, such date is the date on which the status of
ftlteznational
depositary authority was acquired.
Form BP/9 (sole page) CBS/9107
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
36
BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Metapontum Agrobios vIAeiLITY STAT~ENT
S.S.IOnlca 106 km. 448,2 issued pursuant to Rule 10.2 by the
7~JO1O METAPONTO MT INTERNATIONAL DEPOSITARY A~)THORITY
identified on the follcwing page
Itaii~
name and acdress of the party tc whom the
viabi_'ity statement is issued
I I. DEPOSITOR I II. IDENTIFICATION OF TF~ MZCROORC,ANISM
Name: MetapOntum AgrOblOS Accession number given by the
INTERNATIONAL DEPOSIT.ARY AUTHORITY:
I
C8S 101642
Address: S.S. lonica 106
km. 448,2
75010 METAPONTO MT Date (dd-mm-yy) of the deposit
or of the
Italie3 t r ans f a r : 1
III. VIABILITY STATEDtENT
The viability of the microorganism
identified under II above
was tested
on 08-03-99 2 On that date(dd-mm-yy),
the
said
microorganism
was
~3
Xu viable
~3
no longer viable
lIndicate the date of the original deposit oz, where a new deposit or a
transfer has been
made, the most recent relevant date (date of the new deposit or date of the
transfer).
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most
recent
viability test.
3
Mark with a cross the applicable box.
Form BP/9 (first page)
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
37
IV. COI~ITIONS BNDER WHICH THE VIABILITY HAS BEEN PERFORI~D
I V. IZ7TERNATIONAL DEPOSITARY ADTHORITY
Name: CentraalbureauvOOrSCtlimmeICUltures Signature (s) of persons) having the
power
to represent the International Cepositary I
A~hc~i~p-ox~of-authorized official(s): I
Address: posterstraat 1 ~ ~' .
PØ Box 273 l
3740 AG BAARN Mrs F.B. Snippe-Claus S pers
The Netherlands Date (dd-mm-y.y) : 11-03- "
9 Fill in if the information has been requested and if the results of the test
were
negative.
Fozm HP/9 (second and last page)
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
1
SEQUENCE LISTING
NUMBER OF SEQUENCES: 5
1
(1) INFORMATION FOR Q ID NO:1:
SE
(i) SEQUENCE CHARACTER ISTICS:
(A) LENGHT: 1216 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Sing le
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: A (genomic)
DN
(xi) SEQUENCE DESCRIPTION:
GAATTCCGCA TGTCTACTGT CATAACCAGC AACAACAGTG 40
AGAAGAAAGG AGGAAGCAGC CACCTTAAGC GAGCGCCGCA 80
CACGAAGCCT CCTTTCACAC TTGGTGACCT CAAGAGAGCC 120
ATCCCACCCC ATTGCTTTGA ACGCTCTTTT GTGCGCTCAT 160
TCTCCTATGT TGCCTATGAT GTCTGCTTAA GTTTTCTTTT 200
CTACTCGATC GCCACCAACT TCTTCCCTTA CATCTCTTCT 240
CCGCTCTCGT ATGTCGCTTG GCTGGTTTAC TGGCTCTTCC 280
AAGGCTGCAT TCTCACTGGT CTTTGGGTCA TCGGCCATGA 320
ATGTGGCCAT CATGCTTTTA GTGAGTATCA GCTGGCTGAT 360
GACATTGTTG GCCTAATTGT CCATTCTGCA CTTCTGGTTC 400
CATATTTTTC ATGGAAATAT AGCCATCGCC GCCACCATTC 440
TAACATAGGA TCTCTCGAGC GAGACGAAGT GTTCGTCCCG 480
AAATCAAAGT CGAAAATTTC ATGGTATTCT AAGTACTTAA 520
ACAACCCGCC AGGTCGAGTT TTGACACTTG CTGCCACGCT 560
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2
CCTCCTTGGC TGGCCTTTAT ACTTAGCTTT CAATGTCTCT 600
GGTAGACCTT ACGATCGCTT TGCTTGCCAT TATGATCCCT 640
ATGGCCCAAT ATTTTCCGAA AGAGAAAGGC TTCAGATTTA 680
CATTGCTGAC CTCGGAATCT TTGCCACAAC GTTTGTGCTT 720
TATCAGGCTA CAATGGCAAA AGGGTTGGCT TGGGTAATGC 760
GTATCTATGG GGTGCCATTG CTTATTGTTA ACTGTTTCCT 800
TGTTATGATC ACATACTTGC AGCACACTCA CCCAGCTATT 840
CCACGCTATG GCTCATCGGA ATGGGATTGG CTCCGGGGAG 880
CAATGGTGAC TGTCGATAGA GATTATGGGG TGTTGAATAA 920
AGTATTCCAT AACATTGCAG ACACTCATGT AGCTCATCAT 960
CTCTTTGCTA CAGTGCCACA TTACCATGCA ATGGAGGCCA 1000
CTAAAGCAAT CAAGCCTATA ATGGGTGAAT ATTACCGGTA 1040
TGATGGTACC CCATTTTACA AGGCATTGTG GAGGGAGGCA 1080
AAGGAGTGCT TGTTCGTCGA GCCAGATGAA GGAGCTCCTA 1120
CACAAGGCGT TTTCTGGTAC CGGAACAAGT ATTAAAP~AAG 1160
TGTCATGTAG CCTGTTTCTT TAAGAGAAGT AATTAGAACA 1200
AGAAGGAATG GTCGAC 1216
(1) INFORM ATION FOR
SEQ ID N0:2:
(i) SEQUEN CE CHARACTERISTICS:
(A) LENGHT : 624 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS:
Single
(D) TOPOLOGY:
Linear
(ii) MOLEC ULE TYPE: rimer
p
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
3
(xi) SEQUENCE
DESCRIPTION:
GAATTCCGCA TGTCTACTGT CATAACCAGC AACAACAGTG 40
AGAAGAAAGG AGGAAGCAGC CACCTTAAGC GAGCGCCGCA 80
CACGAAGCCT CCTTTCACAC TTGGTGACCT CAAGAGAGCC 120
ATCCCACCCC ATTGCTTTGA ACGCTCTTTT GTGCGCTCAT 160
TCTCCTATGT TGCCTATGAT GTCTGCTTAA GTTTTATTTT 200
CTACTCGATC GCCACCAACT TCTTCCCTTA CATCTCTTCT 240
CCGCTCTCGT ATGTCGCTTG GCTGGTTTAC TGGCTCTTCC 280
AAGGCTGCAT TCTCACTGGT CTTTGGGTCA TCGGCCATGA 320
ATGTGGCCAT CATGCTTTTA GTGAGTATCA GCTGGCTGAT 360
GACATTGTTG GCCTAATTGT CCATTCTGCA CTTCTGGTTC 400
CATATTTTTC ATGGAAATAT AGCCATCGCC GCCACCATTC 440
TAACATAGGA TCTCTCGAGC GAGACGAAGT GTTCGTCCCG 480
AAATCAAAGT CGAAAATTTC ATGGTATTCT AAGTACTTAA 520
ACAACCCGCC AGGTCGAGTT TTGACACTTG CTGCCACGCT 560
CCYCCYYHHC TGGCCTTTAT ACTTAGCTTT CAATGTCTCT 600
GGTAGACCTT ACGATCGCGT CGAC 624
(1) INFORMATION EQ ID N0:3:
FOR S
(i) SEQUEN CE CHARACTERISTICS:
(A) LENGHT : 633 base pairs
(B) TYPE: Nucleic d
aci
(C) STRAND EDNESS: gle
Sin
(D) TOPOLO GY: Linear
(ii) MOLEC ULE TYPE: )
DNA (genomic
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
4
(xi) SEQUENCE
DESCRIPTION:
GAATTCAATG TCTCTGGTAG ACCTTACGAT CGCTTTGCTT 40
GCCATTATGA TCCCTATGGC CCAATATTTT CCGAAAGAGA 80
AAGGCTTCAG ATTTACATTG CTGACCTCGG AATCTTTGCC 120
ACAACGTTTG TGCTTTATCA GGCTACAATG GCAAAAGGGT 160
TGGCTTGGGT AATGCGTATC TATGGGGTGC CATTGCTTAT 200
TGTTAACTGT TTCCTTGTTA GGATCACATA CTTGCAGCAC 240
ACTCSCCCAG CTATTCCACG CTATGGCTCA TCGGAATGGG 280
ATTGGCTCCG GGGAGCAATG GTGACTGTCG ATAGAGATTA 320
TGGGGTGTTG AATAAAGTAT TCCATAACAT TGCAGACACT 360
CATGTAGCTC ATCATCTCTT TGCTACAGTG CCACATTACC 400
ATGCAATGGA GGCCACTAAA GCAATCAAGC CTATAATGGG 440
TGAATATTAC CGGTATGATG GTACCCCATT TTANCAAGGC 480
ATTGTGGAGG GAGGCAAAGG AGTGCTTGTT CCGTCGAGCC 520
AGATGAAGGA GCTCNTACAC AAGGCGTTTT CTGGTACCGG 560
AACAAGTATT AAAAAAGTGT CATGTAGCCT GTTTCTTTAA 600
GAGAAGTAAT TAGAACAAGA AGGAATGGTC GAC 633
(1) INFORMATION
FOR SEQ
ID N0:4:
(i) SEQUEN CE CHARACTERISTICS:
(A) LENGHT : 762 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS:
Single
(D) TOPOLOGY:
Linear
(ii) MOLEC ULE TYPE:
cDNA
CA 02374015 2001-11-15
WO 00/70052 PCT/EP00/04181
(xi) SEQUENCE
DESCRIPTION:
TCTAGAATCC TTTCTTTCTT 40
ATCTCTCTCA
GTTTTCTTTC
TCTTTTCTTT CTCAA 81
GTTTATTTAT ATG
TTTTCTTGCT GCA
Met
Ala
5 1
GAA ACA CCA TCA AAG CGC CAA AGG AGT AAT GAA ACT 117
Glu Thr Pro Ser Lys Arg Gln Arg Ser Asn Glu Thr
5 10
TAT GCA GAG GAT GTC GAA GAA AGT AAG AGA CAT AAA 153
Tyr Ala Glu Asp Val Glu Leu Leu Leu Leu Leu Leu
20 25
TCT TAC AAC CAA ATA CTC TCC CTT CTT GAA GAA GAA 189
Pro Thr Gln Asp Ile Ser Ser Leu Ile Leu Leu Leu
30 35
15 GAA GAA GAG CCA ACC CAA GAC ATC TCT TCT CTA ATA 225
Leu Leu Leu Pro Thr Gln Asp Ile Ser Ser Leu Ile
40 45 50
ACT ACA CTT CAG CAA GAA CTC GAC GAC CCT CTT TCT 261
Thr Thr Leu Gln Gln Glu Leu Asp Asp Pro Leu Ser
55 60
TGT CCA TCG ACA GAA ACT GGC AGA GAG AGC CCT TTT 297
Cys Pro Ser Thr Glu Thr Gly Arg Glu Ser Pro Phe
65 70
GCT ACA ATC ACG TCC GCC ATT GAA GAC TAC CCA TCT 333
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6
Ala Thr Ile Thr Ser Ala Ile Glu Asp Tyr Pro Ser
75 80 85
TCC TCT TCA TCA TCATCT TCT TCT TCT TCT TCC ATG 369
Ser Ser Ser Ser SerSer Ser Ser Ser Ser Ser Met
90 95
TTA TTG AAG GAA GACGAG GAA GAT GAT AAA GAT AGG 405
Leu Leu Lys Glu AspGlu Glu Asp Asp Lys Asp Arg
100 105 110
GTT ATA AGA CAT CTTCTT GAA GCC TCT GAT GAT GAA 441
Val Ile Arg His LeuLeu Glu Ala Ser Asp Asp Glu
115 120
CTT GGG ATT CCA AATACA GAA ACT GGT TCT GGT TTC 477
Leu Gly Ile Pro AspThr Glu Thr Gly Ser Gly Phe
125 130
GAT GAT GGC TAT GAAGGT TTT GTT AGT AGT GCA AAC 513
Asp Asp Gly Tyr GluGly Phe Val Ser Ser Ala Asn
135 140 145
GGG TTT AGT GGA GTAGAT GGA TTC TCT CTT TGT GAT 549
Gly Phe Ser Gly ValAsp Gly Phe Ser Leu Cys Asp
150 155
GGC TTA TGG GAG ATTGAA GAT GCT AAT GCT AAC TAC 585
Gly Leu Trp Glu IleGlu Asp Ala Asn Ala Asn Tyr
160 165 170
TAT GCC CTG TTG CAATCT GAA CTA TTC ATG TAG CTA 621
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7
Tyr Ala Leu Leu Gln Ser Glu Leu Ser
Phe Met Leu
175 180
TAAT CTTTAA AAACAAAAAA AAAGGAAAAA GAAAAGGAAA 661
GAAAAAGGGA AGAAACGAAA 701
TTAGGGGGTG
TAGATGGAAA
TGAT AGAGCT TCAAAAGATG AAAAAAGAAA AGAATTCGTG 741
GGAT ATAAAA TGGAGGAGCT C 762
(1) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 180 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION:
Met Ala Glu Thr Pro Ser Lys Arg Arg Ser Glu Thr Tyr
Gln Asn
1 5 10 15
Ala Glu Asp Val Glu Leu Leu Leu Leu Leu Thr Gln Asp
Leu Pro
20 25 30
Ile Ser Ser Leu Ile Leu Leu Leu Leu Leu Thr Gln Asp
Leu Pro
35 40 45
Ile Ser Ser Leu Ile Thr Thr Leu Gln Glu Asp Asp Pro
Gln Leu
50 55 60
Leu Ser Cys Pro Ser Thr Glu Thr Arg Glu Pro Phe Ala
Gly Ser
65 70 75
Thr Ile Thr Ser Ala Ile Glu Asp Pro Ser Ser Ser Ser
Tyr Ser
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80 85 90
Ser Ser Ser Ser Ser Ser Ser Met Leu Leu Lys Glu Asp Glu Glu
95 100 105
Asp Asp Lys Asp Arg Val Ile Arg His Leu Leu Glu Ala Ser Asp
110 115 120
Asp Glu Leu Gly Ile Pro Asp Thr Thr Ser Gly Phe
Glu Gly Asp
125 130 135
Asp Gly Tyr Glu Gly Phe Val Ser Ala Gly Phe Ser
Ser Asn Gly
140 145 150
Val Asp Gly Phe Ser Leu Cys Asp Leu Glu Ile Glu
Gly Trp Asp
155 160 165
Ala Asn Ala Asn Tyr Tyr Ala Leu Gln Glu Leu Phe
Leu Ser Met
170 175 180
(1) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 22 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii ) MOLECULE TYPE: primer
(xi ) SEQUENCE DESCRIPTION:
GGATCCCTCA GGAAAGTGCT TA
(1) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGHT: 26 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
TCTAGACATT CCTTCTTGTT CTAATT
(1) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 21 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
GAATTCCGCA TGTCTACTGT C
(1) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 21 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
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WO 00/70052 PCT/EP00/04181
GTCGACCATT CCTTCTTGTT C
(1) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 18 base pairs
5 (B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
10 GTCGACGCGA TCGTAAGG
(1) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 22 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
GAATTCAATG TCTCTGGTAG AC
(1) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 21 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
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(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
AGGGATGTTT AATACCACTA C
(1) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 21 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
GCACAGTTGA AGTGAACTTG C
(1) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGHT: 21 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
GTGCGACATC ATCATCGGAA G
(1) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGHT: 21 base pairs
(B) TYPE: Nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: primer
(xi) SEQUENCE DESCRIPTION:
CCTAAGAGTC ACTTTAAAAT T
