U.S. patent application number 10/234026 was filed with the patent office on 2003-05-22 for production of ascorbic acid in plants.
Invention is credited to Bauw, Guy Jerome Corneel, Davey, Mark William, Montagu, Marc Charles Ernest Van, Ostergaard, Jens.
Application Number | 20030097679 10/234026 |
Document ID | / |
Family ID | 19764934 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030097679 |
Kind Code |
A1 |
Bauw, Guy Jerome Corneel ;
et al. |
May 22, 2003 |
Production of ascorbic acid in plants
Abstract
The present invention relates to a polynucleotide in isolated
form, which polynucleotide codes for a protein with the activity of
the enzyme L-galactono-.gamma.-lactone dehydrogenase, which
polynucleotide comprises at least the L-galactono-.gamma.-lactone
dehydrogenase activity-determining parts of the coding part of the
sequence or a sequence derived therefrom on the basis of the
degeneration of the genetic code. The invention further relates to
the use of the polynucleotide in the production of transgenic
plants, plant cells, or other eukaryotic cells.
Inventors: |
Bauw, Guy Jerome Corneel;
(Proven-Poperinge, BE) ; Davey, Mark William;
(Gent, BE) ; Ostergaard, Jens; (Lyngby, DK)
; Montagu, Marc Charles Ernest Van; (Brussel,
BE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
19764934 |
Appl. No.: |
10/234026 |
Filed: |
August 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10234026 |
Aug 29, 2002 |
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09423468 |
Feb 15, 2000 |
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6469149 |
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09423468 |
Feb 15, 2000 |
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PCT/EP98/02830 |
May 7, 1998 |
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Current U.S.
Class: |
800/278 ;
435/190; 435/320.1; 435/419; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12P 17/04 20130101;
C12N 15/8243 20130101; C12N 9/001 20130101; C12N 15/8271
20130101 |
Class at
Publication: |
800/278 ;
536/23.2; 435/69.1; 435/190; 435/419; 435/320.1 |
International
Class: |
A01H 001/00; C07H
021/04; C12N 005/04; C12N 009/04; C12P 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 7, 1997 |
NL |
1006000 |
Claims
1. Polynucleotide in isolated form, which polynucleotide codes for
a protein with the activity of the enzyme
L-galactono-.gamma.-lactone dehydrogenase, which polynucleotide
comprises at least the L-galactono-.gamma.-lactone dehydrogenase
activity-determining parts of the coding part of the nucleotide
sequence, which is shown in FIG. 3, or a sequence derived therefrom
on the basis of the degeneration of the genetic code.
2. Polynucleotide as claimed in claim 1, which polynucleotide is a
cDNA which codes for the enzyme L-galactono-.gamma.-lactone
dehydrogenase and at least substantially comprises the coding part
of the nucleotide sequence which is shown in FIG. 3.
3. Polynucleotide as claimed in claim 1 or 2 for use in the
production of transgenic plant cells, plant tissues or plants with
an increased content of the enzyme L-galactono-.gamma.-lactone
dehydrogenase relative to non-transgenic plant cells, plant tissues
or plants.
4. Polynucleotide as claimed in claim 1 or 2 for use in the
production of transgenic plant cells, plant tissues or plants with
an increased content of ascorbic acid relative to non-transgenic
plant cells, plant tissues or plants.
5. Polynucleotide as claimed in claim 1 or 2 for use in the
transformation and/or transfection of eukaryotic cells in order to
bring about expression of the polynucleotide therein.
6. Transgenic plant cells which carry in their genome a
polynucleotide as claimed in claim 1 or 2 not naturally present
therein.
7. Transgenic plant cells as claimed in claim 6 which form part of
a transgenic plant tissue and/or a tranagenic plant.
8. Transgenic plant tissue consisting at least partially of
transgenic plant cells as claimed in claim 6.
9. Transgenic plant tissue as claimed in claim 8 which forms part
of a transgenic plant.
10. Transgenic plant which consists at least partially of plant
cells as claimed in claim 6.
11. Transgenic plant as claimed in claim 10, obtainable by
transforming a plant cell with a polynucleotide as claimed in claim
1 or 2 and by regenerating a plant from the transformed plant
cell.
12. Transgenic plant as claimed in claim 10 or 11, characterized in
that the plant is thale cress (Arabidopsis thaliana), tobacco
(Nicotiana tabacum), tomato, potato or corn.
13. Transformed and/or transfected eukaryotic cell, comprising in
its genome a polynucleotide as claimed in claim 1 or 2.
14. Recombinant L-galactono-.gamma.-lactone dehydrogenase,
obtainable by expression of a polynucleotide as claimed in claim 1
or 2 in a suitable host.
15. Recombinant L-galactono-.gamma.-lactone dehydrogenase as
claimed in claim 14 which is isolated from a transgenic plant
tissue as claimed in claim 7 or 8, a transgenic plant as claimed in
claim 9 or 10, or a eukaryotic cell as claimed in claim 13.
16. Transformation system, comprising a transformation vector or
set of vectors, at least one of which includes a nucleotide
sequence which codes for the enzyme L-galactono-.gamma.-lactone
dehydrogenase.
17. Transformation system as claimed in claim 15, comprising
Agrobacterium and a binary vector comprising a polynucleotide as
claimed in claim 1 or 2.
18. Use of polynucleotide as claimed in claim 1 or 2 and/or the
transformation system as claimed in claim 16 or 17 for producing a
transgenic plant or plant tissue with an increased content of
L-galactono-.gamma.-lactone dehydrogenase compared with a
non-transgenic plant or plant tissue.
19. Use of the polynucleotide as claimed in claim 1 or 2 and/or the
transformation system as claimed in claim 16 or 17 for producing a
transgenic plant or plant tissue with an increased ascorbic acid
content compared to a non-transgenic plant or plant tissue.
20. Use as claimed in claim 17 or 18, wherein the plant is thale
cress (Arabidopsis thaliana), tobacco (Nicotiana tabacum), tomato,
potato or corn.
21. Use of a polynucleotide as claimed in claim 1 or 2 for
transfecting and/or transforming a eukaryotic cell.
22. Method for producing plants or plant tissues with an increased
ascorbic acid content, comprising of transformation of a plant cell
with a gene construct which comprises at least the polynucleotide
as claimed in claim 1 or 2, optionally in the presence of suitable
transcription and/or translation regulation factors, and
regeneration of a transgenic plant or plant tissue from the plant
cell.
23. Method for producing the enzyme L-galactono-.gamma.-lactone
dehydrogenase, comprising of transfecting and/or transforming a
eukaryotic cell with a gene construct which comprises at least the
polynucleotide as claimed in claim 1 or 2, optionally in the
presence of suitable transcription and/or translation regulation
factors, expressing the enzyme in the transfected and/or
transformed cell and optionally isolating the enzyme from the cell
and/or its culture medium.
24. Gene construct comprising a polynucleotide as claimed in claim
1 or 2, optionally in the presence of transcription and/or
translation regulation factors.
25. Gene construct as claimed in claim 24, further comprising
targeting sequence for targeting the encoded enzyme to various
parts of the plant cell.
26. Gene construct as claimed in claim 25, wherein the parts of the
plant cell are the cytoplasm, vacuoles, chloroplasts, mitochondria,
lysosomes, endoplasmatic reticulum, Golgi apparatus.
27. Method for purifying the enzyme L-galactono-.gamma.-lactone
dehydrogenase, comprising of: a) passing a protein extract of
cauliflower florets through an ion-exchange column: b) collecting a
number of fractions eluting from the column and determining the
GLDase activity of the fractions; c) combining the fractions with
GLDase activity and passing them through a Phenyl Sepharose CL 4B
column; d) collecting a number of fractions eluting from the column
and determining the GLDase activity of the fractions; e) combining
the fractions with GLDase activity and passing them through a gel
filtration column; f) collecting a number of fractions eluting from
the column and determining the GLDase activity of the fractions; g)
combining the fractions with GLDase activity and passing them
through an FPLC Resource Q-column; h) collecting a number of
fractions eluting from the column and determining the GLDase
activity of the fractions; i) combining the fractions with GLDase
activity and passing them through a FPLC Poros 20 SP-column; j)
collecting a number of fractions eluting from the column and
determining the GLDase activity of the fractions.
28. Method for increasing the L-ascorbic acid levels in plants,
comprising: a) provision of plants that have been transformed with
the sense version of the GLDase gene, and b) providing the said
plants with the precursor L-galactono-.gamma.-lactone in order to
induce increased L-ascorbic acid synthesis.
29. Transgenic plants having in their genome an antisense version
of the GLDase gene resulting in a decreased amount of ascorbic acid
as compared to non-transgenic plants for use a model system or
biosensor for oxidative stress.
Description
[0001] The present invention relates to a polynucleotide, in
particular a CDNA, which codes for L-galactono-.gamma.-lactone
dehydrogenase (GLDase), an enzyme involved in the biosynthesis of
ascorbic acid (vitamin C) in plants. The invention further relates
to the use of this cDNA for the synthesis of the enzyme and for the
production of transgenic plant and animal cells, plant tissues and
plants producing the enzyme.
[0002] Ascorbic acid is synthesized in all higher plants and in
almost all higher animals, with the exception of humans and other
primates, the guinea pig and a number of birds. Opinions differ
concerning the presence of ascorbic acid in micro-organisms. It
appears to be present in a number of yeasts, although there are
also reports which suggest that ascorbic acid analogues are found
in micro-organisms.
[0003] In the animal and plant kingdom, ascorbic acid is formed by
different routes. In animals, glucose is the primary precursor for
the biosynthesis of ascorbic acid, and the last step in the
biosynthetic pathway is catalyzed by a microsomal enzyme:
L-gulono-.gamma.-lactone oxidase This enzyme has already been
isolated from rat, goat and chicken liver and kidney tissues.
[0004] The pathway of ascorbic acid biosynthesis in plants,
however, is not yet entirely clear, but there are indications that
at least two different biosynthetic pathways exist. Isherwood et
al., Biochem. J. 56:1-15 (1954) postulated that the biosynthesis of
ascorbic acid starting from D-galactose proceeds via
L-galactono-.gamma.-lactone to L-ascorbic acid. Mapson et al.,
Biochem. J. 56:21-28 (1954) were the first to study this oxidation
of L-galactono-.gamma.-lactone to ascorbic acid, a reaction which
is catalyzed by L-galactono-.gamma.-lactone dehydrogenase.
[0005] The presence of L-galactono-.gamma.-lactone dehydrogenase
activity has been described for different plants, including pea,
cabbage and potato. ba et al., J. Biochem. 117:120-124 (1995) have
recently purified the enzyme activity from sweet potato tubers.
[0006] Distinct from this biosynthetic pathway, however, an
alternative pathway has been proposed which takes as starting point
the conversion of D-glucose, and proceeds via L-glucosone and
L-sorbosone to ascorbic acid. An NADP-dependent dehydrogenase,
which catalyses the conversion of L-sorbosone to ascorbic acid, has
been partially purified from bean and spinach leaves (Loewees et
al., Plant Physiol. 94:1492-1495 (1990)).
[0007] The primary function of ascorbate is as a reducing agent.
This is universal. Ascorbic acid is also important as a cofactor
for certain enzymatic reactions, including the production of
collagen in vertebrates. Since humans are completely dependent on
ingested food for the acquisition of ascorbate, it is desirable to
increase the vitamin C content of plants and fruit.
[0008] Owing to its reducing activity, vitamin C plays a role in
the protection of plants and animals against environmental stresses
including heat, cold, drought, oxidative stress etcetera. Less
stress-sensitive or even stress-resistant plants can therefore play
an important part in the economy and agriculture of the world.
[0009] It is the object of the present invention to create the
possibility of genetically modifying plants such that they contain
an increased content of ascorbic acid relative to non-modified
plants.
[0010] For this purpose the invention provides a polynucleotide in
isolated form, which polynucleotide codes for a protein with the
activity of the enzyme L-galactono-.gamma.-lactone dehydrogenase,
which polynucleotide comprises at least the
L-galactono-.gamma.-lactone dehydrogenase activity-determining
parts of the coding part of the nucleotide sequence, which is shown
in FIG. 3, or a sequence derived therefrom on the basis of the
degeneration of the genetic code. The invention is of course not
limited to polynucleotides with exactly the same sequence as that
shown in FIG. 3. It will be apparent to the molecular biologist
skilled in the techniques that a certain degree of modification of
the sequence shown in FIG. 3 is permitted while still falling
within the scope of the claim. The polynucleotide is for instance
the CDNA shown in FIG. 3.
[0011] Polynucleotides according to the invention can be used in
the production of transgenic plant and animal cells, plant tissues
or plants with an increased content of the enzyme
L-galactono-.gamma.-lactone dehydrogenase relative to
non-transgenic plant cells, plant tissues or plants. Such an
increased concentration of GLDase will result in plant cells, plant
tissues or plants with an increased content of ascorbic acid and
with an increased capacity for biosynthesis relative to
non-transgenic plant cells, plant tissues or plants.
[0012] Plants which can advantageously be used for transformation
with the polynucleotide according to the invention are for instance
thale cress (Arabidopsis thaliana), tobacco (Nicotiana tabacum),
tomato, potato, or corn, without this list being limitative.
[0013] Polynucleotides according to the invention can likewise be
expressed in eukaryotic cells, such as yeast cells or mammalian
cells, in particular fibrosarcoma cells.
[0014] The invention further relates to a recombinant
L-galactono-.gamma.-lactone dehydrogenase which can be obtained by
expression of a polynucleotide according to the invention in a
suitable host. The recombinant L-galactono-.gamma.-lactone
dehydrogenase can be isolated from transgenic plant tissues or
transgenic plants, but also from yeasts or from animal cells.
[0015] The invention also relates to a transformation system,
comprising a transformation vector or set of vectors, at least one
of which includes a nucleotide sequence which codes for the enzyme
L-galactono-.gamma.-lacton- e dehydrogenase. The transformation
system preferably comprises Agrobacterium and a binary vector.
[0016] Plants or plant tissues with an increased ascorbic acid
content can be produced by transforming a plant cell with a gene
construct comprising at least the polynucleotide specified in the
invention, optionally linked to targeting sequences for specific
organelles, and/or in the presence of suitable transcription and/or
translation regulation factors, and regenerating from the plant
cell a transgenic plant or plant tissue. The gene construct with
the polynucleotide according to the invention can optionally be
combined with other genes coding for enzymes which can interfere in
the ascorbic acid synthesis, such as L-sorbosone dehydrogenase,
UDP-glucuronic acid epimerase, D-galacturonic acid dehydrogenase
and ascorbate-regulating enzymes, which may determine the rate of
ascorbic acid synthesis.
[0017] The enzyme may ultimately be targeted to a particular part
of the plant cell, such as the cytoplasm, vacuoles, chloroplasts,
mitochondria, lysosomes, endoplasmatic reticulum, Golgi
apparatus.
[0018] Eukaryotic cells expressing the enzyme GLDase can be
obtained by transfection with the polynucleotide according to the
invention.
[0019] Finally, the invention relates to a new method for purifying
the enzyme L-galactono-.gamma.-lactone dehydrogenase. This method
comprises of passing a protein extract of cauliflower florets
through an ion exchange column: collecting a number fractions
eluting from the column and determining the GLDase activity of the
fractions; combining fractions with GLDase activity and passing
thereof through a Phenyl Sepharose CL 4B column; collecting the
column eluate in a number of fractions and determining the GLDase
activity of the fractions; combining those fractions with GLDase
activity and passing thereof through a gel filtration column;
collecting a number of fractions eluting from the column and
determining the GLDase activity of the fractions; combining the
fractions with GLDase activity and passing through an FPLC Resource
Q-column; collecting a number of fractions eluting from the column
and determining the GLDase activity of the fractions; combining the
fractions with GLDase activity and passing thereof over an FPLC
Poros 20 SP-column; collecting a number of fractions eluting from
the column and determining the GLDase activity of the fractions.
The enzyme purified by us is lycorine-insensitive, in contrast to
the literature which states that L-galactono-.gamma.-lactone
dehydrogenase is inhibited by lycorine (De Tullio et al., Boll.
Soc. Ital. Biol. Sper. 70:57-62 (1994); Arrigoni et al., Boll. Soc.
Ital. Biol. Sper. 72:37-43 (1996)).
[0020] Furthermore, the invention provides for a method for
increasing the L-ascorbic acid levels in plants, comprising:
[0021] a) provision of plants that have been transformed with the
sense version of the GLDase gene, and
[0022] b) providing the said plants with the precursor
L-galactono-.gamma.-lactone in order to induce increased L-ascorbic
acid synthesis.
[0023] According to another aspect thereof the invention provides
transgenic plants having in their genome an antisense version of
the GLDase gene resulting in a decreased amount of ascorbic acid as
compared to non-transgenic plants for use a model system or
biosensor for oxidative stress.
[0024] The present invention will be elucidated with reference to
the non-limitative examples provided below.
EXAMPLES
Example 1
[0025] Purification of L-galactono-.gamma.-lactone
dehydrogenase
[0026] 1. Introduction
[0027] Using a 5-step purification method which has not previously
been described, an acceptable yield of the enzyme
L-galactono-.gamma.-lactone dehydrogenase (further designated
GLDase) was obtained.
[0028] 2. Materials and Methods
[0029] 2.1. Materials
[0030] Sephacryl SF-200, DEAE Sepharose and Phenyl Sepharose CL-4B
were obtained from Pharmacia, Sweden. L-galactono-.gamma.-lactone,
D-galactono-.gamma.-lactone, D-gulono-.gamma.-lactone,
L-gulono-.gamma.-lactone, L-mannono-.gamma.-lactone, D-galactonic
acid, D-glucuronic acid, D-gluconic acid and
P-hydroxymercuribenzoic acid were from Sigma Chemical, USA.
D-erythronic lactone, D-xylonic lactone and N-ethyl-maleimide were
purchased from Aldrich Chemical Company, USA. Restriction enzymes
were from Pharmacia, Sweden and (.alpha.-.sup.32P) dCTP was from
Amersham Corp., USA. The cauliflowers (Brassica olecera var.
botrytis) were obtained from a field near Ghent and stored at
4.degree. C. until use
[0031] 2.2. Preparation of an Extract
[0032] Cauliflower florets (7.5 kg) were cut into small pieces,
weighed and homogenized in a pre-cooled blender in ice-cold buffer
A (400 mM sucrose, 100 mM sodium phosphate buffer, pH 7.4) (1 l/kg
fresh weight). The homogenate was pressed through four layers of
Miracloth tissue (Calbiochem-Novabiochem Corp., La Jolla, Calif.,
USA) and centrifuged for 45 minutes at 13,500.times.g in a GS3
rotor (Sorvall). The pellet containing the mitochondria (about 250
g material) was kept at -70.degree. C. until use.
[0033] Before use the pellet was slowly defrosted in a microwave
oven and resuspended in 1/10 vol. (750 ml) buffer A. Cold acetone
(-20.degree. C.) was added slowly while stirring (10.times.vol.).
The mixture stood for 30 minutes at 4.degree. C. The precipitated
protein was collected by filtration through prefilter paper (A15,
Millipore, Bedford, USA) and resuspended in 1/10 vol. buffer B (40
mM Tris-HCl, pH 9.0) followed by 5 hours of dialysis against 10
volumes buffer B. The denatured proteins were removed by
centrifugation (10,000.times.g for 15 minutes). GLDase was purified
from the supernatant, further designated as "protein extract",
using the purification procedure described below.
[0034] All operations relating to preparation of the extract and
enzyme purification were performed at 4.degree. C. unless otherwise
stated.
[0035] 2.3. Enzyme Purification
[0036] The protein extract was placed on a DEAE Sepharose column
(5.times.12 cm) equilibrated with buffer B. After washing with 4
volumes of buffer B at a flow rate of 60 ml per hour, the elution
was carried out with 0.5 M NaCl in the same buffer. Fractions of 8
ml were collected at a flow rate of 60 ml per hour.
[0037] The GLDase activity of the fractions was determined
spectrophotometrically by monitoring the
L-galactono-.gamma.-lactone dehydrogenase-dependent reduction of
cytochrome c at 22.degree. C. A typical reaction mixture contained
the enzyme extract, 1.5 mg/ml cytochrome c and 4.2 mM
L-galactono-.gamma.-lactone in 0.05 M Tris-HCl buffer (pH 8.4).
Reduction of cytochrome c was monitored by determining the
absorption increase at 550 nm. Under these conditions the speed of
the reaction was linear in respect of time for an initial period of
15 minutes. One unit of enzyme activity was defined as the quantity
of enzyme reducing 2 .mu.mol of cytochrome C per minute.
[0038] The fractions containing GLDase activity were pooled and
ammonium sulphate was added up to a concentration of 1 M. Hereafter
the extract was loaded onto a Phenyl Sepharose CL 4B column
(2.2.times.15.0 cm) which was equilibrated with buffer C (1 M
ammonium sulphate, 25 mM sodium phosphate, pH 7.0). After washing
with two volumes of buffer C the elution was carried out with a
linear gradient of 0-80% ethylene glycol in 25 mM sodium phosphate,
pH 7.0, at a flow rate of 30 ml/hour.
[0039] The GLDase activity of the fractions was again determined
and GLDase-containing fractions were collected, concentrated to 10
ml by ultrafiltration using a PM 10 membrane (Amicon Corp.) and
loaded onto a Sephacryl SF-200 gel filtration column (2.6.times.94
cm) equilibrated in buffer D (20% ethylene glycol, 40 mM NaCl, 80
mM sodium phosphate, pH 7.4). The enzyme was eluted with the same
buffer at a flow rate of 25 ml per hour. Fractions of 5 ml were
collected and fractions containing activity were combined. It was
possible to keep the gel filtration preparation at 4.degree. C. for
several weeks without loss of activity.
[0040] Two gel filtration preparations were pooled. The
preparations were concentrated and the buffer was replaced by
buffer E (20% ethylene glycol, 20 mM Tris-HCl, pH 8.0) by means of
ultrafiltration. The resulting enzyme solution was loaded onto a 6
ml Resource Q column (Pharmacia) which was equilibrated beforehand
with buffer E and coupled to an FPLC system (Pharmacia). The flow
rate was 1 ml per minute. Elution was carried out with a gradient
of 0 to 450 mM NaCl as follows: 0 to 85 mM in 18 minutes, 85 to 110
mM in 10 minutes, 110 to 130 mM in 14 minutes and 130 to 450 mM in
10 minutes. Fractions of 1 ml were collected. The activity of the
main peak, which eluted at 120 mM NaCl, was pooled and brought to
pH 6 with 50 mM sodium phosphate.
[0041] The pooled fractions were loaded onto a Poros 20 SP column
(Pharmacia) coupled to an FPLC and equilibrated in buffer F (20 mM
sodium phosphate, pH 6.0, 20% ethylene glycol) at a flow rate of 1
ml/minute. The elution was carried out with a gradient of 0 to 500
mM NaCl in buffer F as follows: 125 to 225 mM in 40 minutes and 225
to 500 mM in 37 minutes. Fractions of 2 ml were collected. Two
peaks with activity eluted: peak I at 210 mM and peak II at 225 mM
NaCl. Peak II was dialyzed against 10 mM sodium phosphate, pH
7.2.
[0042] A Zorbax gel filtration column (9.4.times.250 mm, Zorbax
Bioseries GF-250) coupled to an HPLC and equilibrated in 750 mM
NaCl, 50 mM sodium phosphate, pH 7.2 was used as final purification
step.
[0043] Table 1 shows a summary of the purification of GLDase from
cauliflower florets. Because the enzymatic activity was most stable
in 20% ethylene glycol, this reagent was included in all buffers
except the buffers which were used in the first purifications steps
with the DEAE Sepharose and Phenyl Sepharose chromatography. After
the DEAE Sepharose step the total GLDase activity increased
slightly, probably due to removal of inhibitory compounds which
were present in the original crude extract. The FPLC Resource step
increased the purification factor from 63 to 900, although the
recovery is only 42% in comparison with the activity present in the
gel-filtered pool. By the subsequent Poros 20 SP column the
activity was separated into two peaks, designated I and II in FIG.
1. The activity from the latter peak was used for further analysis.
Table 1 shows that GLDase was 1693 times more purified from the
mitochondrial fraction with a recovery of 1.1%. The purity of the
enzyme was tested by means of analytical SDS polyacrylamide gel
electrophoresis (SDS PAGE) in slab gels of 10% polyacrylamide as
according to Chua, Methods Enzymol. 69:434-446 (1980). Proteins
were visualized either by means of Coomassie Brilliant Blue R250
staining (Chua (1980), supra) or silver nitrate staining (Merril et
al., Methods Enzymol. 104:441-447 (1984)). Three polypeptide bands
were found with molecular masses of about 56 kDa, 30 kDa and 26 kDa
(see FIG. 2).
[0044] A partial amino acid sequence was determined as follows.
Purified GLDase from the Porous 20 SP purification step was
separated by means of SDS-PAGE. The proteins were blotted onto
polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford,
USA) as described by Bauw et al., Proc. Natl. Acad. Sci. USA
4806-4810 (1987) with 50 mM Tris/50 mM boric acid (pH 8.3) as
transfer buffer. The transfer was carried out for at least 8 hours
at 35 Volts with a Bio-Rad Transblot apparatus. PVDF membrane-bound
polypeptides were visualized by staining with 0.1% Amido black
solution. The polypeptide bands were excised and a trypsin
digestion was performed in situ, followed by reversed phase HPLC
separation of the generated peptides, as previously described by
Bauw et al., Proc. Natl. Acad. Sci. USA 86:7701-7705 (1989).
Partial amino acid sequence determination by Edman degradation was
carried out on an Applied Biosystems model 473A protein sequencer
in accordance with the instructions of the manufacturer.
[0045] Table 2 shows the sequences of a number of peptides derived
from the GLDase. This shows inter alia that the two low-molecular
bands are dissociation products of the 56 kDa band. The NH.sub.2
terminal sequences of the 56 kDa and the 30 kDa polypeptide bands
are identical.
Example 2
[0046] Sensitivity to Lycorine
[0047] The literature states that lycorine, a pyrrole
phenanthridine alkaloid present in different plants of the
Amaryllidaceae, inhibits the ascorbic acid synthesis at
concentrations from 1 .mu.M. It has recently been demonstrated that
the inhibition of lycorine is based on an interaction with the
enzyme L-galactono-.gamma.-lactone dehydrogenase (De Tullio et al.,
(1994), sunra; Arrigoni et al., (1996), supra).
[0048] Lycorine was isolated from the plant Crinum asiaticum and
the identity of the isolated product was verified by NMR, electron
spray mass spectrometry HPLC analysis and capillary
electrophoresis. Fractions of L-galactono-.gamma.-lactone
dehydrogenase activity isolated from the gel filtration column were
tested for their activity in two different buffers in the presence
of 5 or 50 .mu.M lycorine (see table 3).
[0049] All data indicate that the isolated GLDase is insensitive to
the inhibitor up to a concentration of 50 .mu.M. Additional tests
did not show a decrease in activity even in 100 .mu.M lycorine. A
pre-incubation of one hour of the enzyme with lycorine did not
influence the enzyme activity.
1TABLE 3 Activity of the enzyme expressed in increase in absorption
at 550 nm/second Concentration lycorine (.mu.M) 0 5 50 75 mM PO4 pH
8 1.882 1.690 1.768 75 mM PO4 pH 7.5 1.385 1.372 1.254 75 mM PO4 pH
7.0 0.980 0.857 0.842 75 mM Tris pH 8.9 5.438 5.199 5.507 75 mM
Tris pH 8.2 6.365 6.400 6.127 75 mM Tris pH 7.4 3.627 3.927
3.743
Example 3
[0050] Isolation of the cDNA
[0051] 300 mg cauliflower florets were ground to a powder in liquid
nitrogen with a pestle and mortar. The powder was suspended in 0.5
ml ice-cold extraction buffer (0.1 M LiCl, 5 mM EDTA, 1% (w/v) SDS
and 0.2 M Tris-HCl, pH 7.5) and extracted twice more with
phenol/CH.sub.3Cl/isoamyl alcohol (25:24:1). The aqueous phase was
adjusted to a final concentration of 3 M LiCl and left on ice for 4
hours. The precipitate was collected by centrifuging for 10 minutes
at 20,000.times.g and the pellet was washed with 1 ml 3 M LiCl and
resuspended in 250 .mu.l H.sub.2O treated with diethyl
pyrocarbonate. The LiCl precipitate was repeated and the pellet
washed and resuspended in 250 .mu.l H.sub.2O treated with diethyl
pyrocarbonate (DEPC). The suspension was centrifuged for 10 minutes
at 20,000.times.g to remove insoluble material. Sodium acetate was
added to an end concentration of 0.3 M followed by addition of 2
volumes ethanol and incubation for 15 minutes at -70.degree. C. The
precipitate was collected by centrifuging for 5 minutes at
20,000.times.g.
[0052] The RNA pellet was washed with 70% ethanol and resuspended
in 25 .mu.l H.sub.2O treated with DEPC. The RNA isolated from
cauliflower florets (4 .mu.g) was used to synthesize the first
strand of cDNA as specified in the instruction manual for
Superscript.TM. Preamplification System for First Strand cDNA
Synthesis of Gibco BRL.
[0053] Degenerated oligonucleotides corresponding with the partial
amino acid sequences as shown in example 1 were designed and
synthesized on an oligonucleotide synthesizer (Applied Biosystems,
Poster City, Calif., USA) and used as primers in PCR reactions. The
peptide sequences designated 1, 4 and 7 in table 2 were used to
design the corresponding coding and complementary oligonucleotides.
First-strand cDNA synthesized from cauliflower florets was used as
a template. The amplification mixture consisted of 130 ng matrix
DNA, PCR buffer (100 mM Tris-HCl, 500 mM KCl, 1.5 mM MgCl.sub.2, pH
8.3), 200-300 ng of each primer, 2.5 mM cNTP and 1 unit Taq
polymerase in a total volume of 50 .mu.l.
[0054] The amplification program consisted of 32 cycles of
denaturation for 1 minute at 94.degree. C., annealing for 1 minute
at SOC and primer extension for 2 minutes at 72.degree. C. The
reaction products were separated on 1% agarose gels, excised and
purified in accordance with the QIAEX handbook of Qiagen GmbH,
Germany. The purified products were cloned into a pGEM-T vector
(Promega, Wis., USA). Of the amplified 250 bp to 400 bp bands which
were subcloned into a pGEM-T vector, a 250 bp fragment, which
contained a nucleotide sequence corresponding to the amino acid
sequence of one of the previously determined internal peptides, was
radioactively labelled and used as probe to screen a cDNA library
of cauliflower. The CDNA library was constructed in .lambda.ZAP II
(Stratagene, La Jolla, Calif., USA) and generously donated by
Professor J. S. Hyams (University, London, UK). Portions of the
CDNA library were plated using Escherichia coli XL-1 Blue-cells on
23.times.23 cm baking plates (Nunc, Roskilde, Denmark) with NZY
agar. About 600,000 plaques from the library were transferred in
duplicate to nylon membranes (HYbond N.sup.+; Amersham Corp., USA).
The membranes were treated in accordance with the instructions of
the manufacturer for plaque blotting. DNA was fixed to the
membranes by radiation with ultraviolet light (UV Stratalinker,
Stratagene, La Jolla, Calif., USA). The membranes were subsequently
incubated with the 250 bp PCR amplified fragment which was labelled
with (.alpha.-.sup.32P) dCTP with a random primed DNA labelling kit
from Boehringer Mannheim, Germany. The membranes were first washed
for 4 hours at 65.degree. C. in a hybridization buffer (1% (w/v) of
bovine serum albumin, 7% (w/v) SDS, 1 mM EDTA and 0.25 M sodium
phosphate, pH 7-2) followed by 20 hours of incubation with the
.sup.32P-labelled probe in the hybridization buffer at 65.degree.
C. The membranes were then rinsed twice for 15 minutes with
2.times.SSC containing and 1% SDS at room temperature and exposed
to X-Omat AR-film (Kodak, CT, USA).
[0055] Different positive clones were found- After in vivo excision
of the Bluescript plasmid followed by digestion with EcoRI and KpnI
the two longest cDNA inserts were found to be approximately 2000 bp
long. Subcloning and sequence determination revealed an
uninterrupted open reading frame of 1803 nucleotides. The open
reading frame contained all the tryptic peptides which had
previously been sequenced, the NH.sub.2 terminal amino acid
sequence, the first ATG codon (startcodon) (at position 56), and
ended with a TAA terminator codon from which it was concluded that
the full length cDNA corresponding to the purified protein had been
isolated.
[0056] FIG. 3 shows the derived amino acid sequences of the 1803 bp
open reading frame which codes for 600 amino acids. A piece of 55
bp is possibly the 5' non-coding region and a piece of 206 bp shows
the 3'non-coding region, including a poly(A)tail. A hexanucleotide
AATAAA consensus signal for polyadenylation is found 20 nucleotides
before the poly(A)tail. The nucleotides coding for the NH.sub.2
terminal amino acid sequence are found 273 bp from the initiator
codon, which indicates that the protein is synthesized as a
preprotein (600 amino acids with a calculated molecular mass of
67,829 Da). The resulting mature protein of 509 amino acids has a
calculated molecular mass of 57,837 Da and a theoretical pI-value
of 6.85. The number of acidic (Glu and Asp) and basic amino acids
(His, Lys and Arg) is respectively 74 and 83. A putative
mitochondrial signal peptide is present.
[0057] DNA sequence determinations were carried out in accordance
with the protocols of US Biochemical Corp. Starting sequences were
obtained with the use of T7 and T3 vector primers. Specific primers
were used to complete the sequences on both strands of cDNA. The
sequence analyses were performed with software from the Genetics
Computer Group (Madison, Wis., USA).
Example 4
[0058] Expression in Yeast
[0059] The GLDase cDNA was expressed in Saccharomyces cerevisiae.
For this purpose the Bluescript vector containing the complete cDNA
was digested with ApaI and KpnI and a 27 bp adaptor containing an
NotI restriction site was ligated in the vector linearized with
ApaI and KpnI. The resulting construct contains two NotI
restriction sites and was cloned in the NotI restriction sites of
the pFL61 vector (Minet et al., Plant J. 2:417-422 (1992)). Yeast
cells of the strain W303A (Mat.alpha., ade 2-1, ura 3-1, his 3-11,
15, trp 1-1, leu 2-3, kan.sup.r) were transformed by means of the
method of Dohmen et al., Yeast 7:691-692 (1991) and plated on
selective 1.5% agar plates (without uracil) with minimal SD medium
(0.2% yeast nitrogen basis (Difco, Detroit, Mich., USA), 0.7%
ammonium sulphate, 2.7% glucose) supplemented with adenine,
tryptophan, leucine at a final concentration of 20 .mu.g/ml, and
histidine at a final concentration of 10 .mu.g/ml. Transformed
cells were transferred to liquid SD medium (as above but without
the agar) and cultured for 3 days at 30.degree. C.
[0060] The GLDase cDNA was introduced both in the sense orientation
and in the antisense orientation relative to the PGK
(phosphoglycerate kinase) promoter and terminator. Non-transformed
and transformed yeasts were grown and extracts were prepared and
tested for GLDase activity. Extracts of yeasts which had been
transformed with a sense-oriented GLDase cDNA displayed a three- to
six-fold increase in specific GLDase activities compared with
extracts from non-transformed yeast and yeast which had been
transformed with antisense-oriented GLDase CDNA. Wild type yeast
has no endogeneous GLDase activity. For determination of protein
levels and GLDase activity, cells were harvested by centrifugation
(18.000 g, 15 min.), washed and resuspended in 50 mM Tris-HCl (pH
8.0) and disrupted in a French press.
Example 5
[0061] Transformation of Arabidopsis and Tobacco
[0062] 1. Introduction
[0063] The GLDase cDNA clone has been used to make sense and
antisense GLDase constructs under control of the 35S cauliflower
Mosaic Virus (CaMV) promoter. Agrobacterium-mediated transformation
has been used to produce transgenic arabidopsis and tobacco plants
with the engineered antisense and sense GLDase constructs in order
to down-regulate or to up-regulate the GLDase transcript,
respectively. Increased GLDase activity was observed in plants
transformed with a sense-orientated GLDase cDNA, whereas the
specific GLDase activity was low in several antisense plant-lines
(see table 4). As a consequence decreased ascorbic acid (AA) levels
were measured in antisense transformed plant-lines (see table
5).
[0064] 2. Materials and Methods
[0065] 2.1. Plasmids and Vectors
[0066] The GLDase cDNA was inserted in both orientations into the
pLBR19 vector (Leple et al. (1992), supra) containing the
cauliflower mosaic virus (CaMV) 35S promoter with a double enhancer
sequence (CaMV 70). The promoter, enhancer and GLDase cDNA were
then cloned into the binary vector pBIN19 (Frisch et al. (1995),
supra), which carries an additional neomycin phosphotransferase
(nptII) gene under control of the CaMV 35S promoter.
[0067] The sense construct was made as follows: the GLDase CDNA
contained in a Bluescript vector was cut with PstI and the
resulting partial GLDase cDNA was cloned into the PstI cloning site
of the pLBR19 vector in the sense orientation, followed by excision
of a SalI-ClaI fragment of this construct. The remaining part of
the GLDase cDNA was then inserted as a XhoI-ClaI fragment,
resulting in a pLBR19 vector containing the complete GLDase cDNA
sequence.
[0068] For the antisense construct the following procedure was
followed: a fragment of the Bluescript inserted GLDase cDNA was
generated by XhoI digestion and inserted into the SalI site of the
pLBR19 vector in antisense orientation. Then a SmaI-NsiI fragment
was excised from this construct and the remaining part of the
GLDase CDNA was inserted as a SmaI-Nsi I fragment resulting in a
pLBR19 vector containing the complete GLDase in antisense
orientation The promoter, enhancer, and GLDase cDNA (sense and
anti-sense orientation) were finally cloned into the KpnI-XbaI site
of the binary vector pBIN19.
[0069] The binary plasmids were then mobilized into Agrobacterium,
strain C58 Rif (pMP90) as described by Zham et al., Mol. Gen.
Genet. 194:188-194 (1984).
[0070] DNA electrophoresis, endonuclease digests, ligation
reactions and Escherichia coli (strain DH5.alpha.) transformations
were performed as according to Sambrook et al. (1989), supra.
[0071] 2.2. Transformation and Regeneration
[0072] MP90 Agrobacterium tumefaciens (strain C58 Rif) were grown
with rifampicin (50 mg/ml), gentamicin (100 mg/l) and kanamycin
(200 mg/l) prepared as described by Bechtold et al. (1993), supra
and used for plant infection.
[0073] 2.3. Arabidopsis
[0074] Arabidopsis thaliana (columbia cultivar) plants were grown
on soil, under standard greenhouse conditions The plants were
transformed by vacuum infiltration as described by Bechtold et al.
(1993), supra.
[0075] 2.4. Tobacco
[0076] Transgenic plants were produced from leaf discs of Nicotiana
tabacum (SRI) following Acrobacterium-mediated transformation as
modified by Thomas et al. (1990), supra. Co-cultivation was for 2-3
days in basal medium (BM) containing 0.5 .mu.M 1-naphtaleneacetic
acid and 2.5 .mu.M 6-benzylaminopurine. Leaf discs were then
transferred to BM supplemented with the phytohormones mentioned
above, and 100 mg/ml kanamycin (Sigma, St. Louis, Mo.) and 500 mg/l
carbenicillin (Sigma). Shoots that formed after 4 weeks were rooted
in phytohormone-free BM containing kanamycin.
[0077] Plants were transferred to soil, grown under standard
greenhouse conditions and self-pollinated. Mature seeds were
collected and selected by germination in the presence of kanamycin
(125 mg/l).
[0078] 2.5. Protein Extraction
[0079] Extracts from plants were prepared by grinding 7 g fresh
tissue in liquid nitrogen. Four volumes of buffer containing 100 mM
sodium phosphate (pH 7.4) containing 400 mM sucrose were added. The
homogenate was squeezed through four layers of Miracloth tissue and
centrifuged at 22,000.times.g for 30 min. The pellet was
resuspended in 5 ml 100 mM sodium phosphate (pH 7.4). Cold acetone
(50 ml, -20.degree. C.) was slowly added under stirring and the
mixture allowed to stand for 30 min. at 4.degree. C. The
precipitated protein was collected by centrifugation
(10,000.times.g for 15 min.). The pellet was dried under vacuum for
30 min. and resuspended in 0.5 ml 40 mM Tris-HCl buffer (pH 8.5).
Insoluble proteins were removed by centrifugation (10,000.times.g
for 15 min.). This preparation was desalted by gelfiltration on
pre-packed NAP-10 (Pharmacia) and used for GLDase activity
assays.
[0080] 2.6. Screening of a Genomic Library Prepared by Arabidonsis
thaliana
[0081] For screening of a genomic library of Arabidopsis thaliana,
the GLDase cDNA was radiolabelled and used as a probe. Five
positive clones were isolated. DNA from the largest of these five
clones was digested with several restriction enzymes and
fractionated on 0.8% (w/v) agarose gel and blotted onto a
Hybond-N.sup.+ membrane (Amersham, USA) as recommended by Amersham.
DNA fragments which hybridized to the GLDase CDNA probe were
subcloned into pbluescript KS(+) (Stratagene, USA) and
sequenced.
[0082] 3. Results
[0083] 3.1. Analysis of Plants
[0084] Transformed plants were found with the positive (sense)
orientation of the GLDase CDNA, and these contained GLDase activity
at 2 to 3-fold higher levels, as compared to control plants. In the
plants transformed with the GLDase cDNA in a negative (anti-sense)
orientation, GLDase activity was approximately 25% of the control
plants.
[0085] The ascorbic acid levels of 28 antisense GLDase plants were
generally lower than the control plants. One plant had 35% AA
content compared to the controls and several other plants have
values around 50%. The AA levels of the sense GLDase plants were
generally higher compared to the controls, with one line attaining
134% of the control.
[0086] 3.2. Isolation of GLDase Gene from Arabidopsis
[0087] By screening a genomic Arabidopsis library a 3117 bp DNA
clone was isolated. Comparison with the GLDase CDNA sequence
isolated from cauliflower indicated that the genomic contained 6
introns. The isolated clone contains 260 bp of the promoter region
up-stream to the first ATG (start) codon. The sequence which
corresponds to the last 260 bp from the 3'-end of the GLDase CDNA
was not found (FIG. 5).
[0088] 4. Conclusions
[0089] The results show the presence of a correctly processed and
biologically active GLDase cDNA in the transgenic tobacco plants.
It was possible to measure increased GLDase activity levels in
plants transformed with GLDase cDNA in the sense orientation.
Furthermore, a decreased GLDase activity was measured in plants
transformed with the corresponding cDNA in the antisense
orientation. In these plants lower ascorbic acid levels were
measured.
[0090] Leaf disc assays did not conclusively show if transformed
plants have changed oxidative stress tolerance.
2TABLE 4 enzyme assays transformed tobacco specific total activity
activity (units/min. .times. plants (units/min.) mg protein) %
control 6.0 1.8 100% sense 5.6 2.1 117% sense 2.2 3.7 206% sense
10.5 3.0 167% antisense 2.6 0.5 28% antisense 1.3 0.4 22%
[0091]
3TABLE 5 Anti-oxidant status of transgenic Nicotiana in nmoles/gram
fresh weight plants L-AA L-DHA total control 1135 132 1267 (100%)
sense 1550 152 1702 (134%) antisense 520 34 554 (44%) L-AA =
ascorbic acid L-DHA = oxidized L-AA (dehydro-ascorbic acid)
Example 6
[0092] Expression in Murine Fibrosarcoma Cells
[0093] 1. Construction of the Eukaryotic Expression Vector
pCAGGS/L-galactono-.gamma.-lactone Dehydrogenase
[0094] pCAGGS is an expression vector which is used for the
efficient expression of genes under the control of the chicken
.beta.-actin/rabbit .beta.-globin hybrid promoter+CMV-IE enhancer
in different mammalian cells (FIG. 4). The plasmid is a gift from
Prof. J. Miyazaki (University or Tokyo, Japan) (Niwa et al., Gene
108:193-200 (1991)).
[0095] The L-galactono-.gamma.-lactone dehydrogenase gene was
isolated after digestion of the Bluescript SK vector with XbaI and
KpnI. The KpnI site was blunted with T4 DNA polymerase and the
XbaI/blunt fragment was cloned into the XbaI/BalI sites of the
pCAGGS vector. The XbaI site of the pCAGGS is situated at the end
of the actual promoter, but the use of this site for cloning a gene
has no effect on the expression efficiency.
[0096] 2. Transfection Procedure: Stable Transfection via DNA
Calcium Phosphate Precipitation Technique
[0097] 2.1. Preparation of the Cells
[0098] 28 hours before transfection, L929sA murine fibrosarcoma
cells are placed in culture at a concentration of 2.10.sup.6 cells
per culture bottle of 75 cm.sup.2. The culture medium used is
Dulbeccols modified essential medium (DMEM) enriched with 5% foetal
calf serum (FCS), 5% newborn calf serum (NCS), 3 mM glutamine and
the antibiotics streptomycin and penicillin. The culture conditions
used are 37.degree. C., 5% CO.sub.2.
[0099] 4 hours before transfection the culture medium is replaced
by 10 ml HEPES-buffered minimum essential medium (MEM-HEPES)
enriched with 10% FCS, 3 mM glutamine and antibiotics.
[0100] 2.2. Preparation of DNA Precipitate
[0101] DNA calcium phosphate precipitate is prepared by adding 30
.mu.g DNA (in 0.25 M CaCl.sub.2/0.125 M HEPES pH 7.05) to the same
volume 2.times. concentrated phosphate/HEPES buffer (0.25 M HEPES,
0.27 M NaCl, 6.7 mM CaCl.sub.2, 1.5 mM Na.sub.2HPO.sub.4). The 30
.mu.g DNA is composed from 19 .mu.g carrier DNA (irrelevant
plastnid DNA)+1 .mu.g DNA of the selection plasmid (pSV2 neoplasmid
carrying the neomycin resistance gene)+10 .mu.g
pCAGGS/L-galactono-.gamma.-lactone dehydrogenase (plasmid with
relevant gene).
[0102] 2.3. Transfection
[0103] The DNA precipitate is placed together with 10 .mu.M
chloroquine on the cells, and the mixture incubated for 4 hours in
5% CO.sub.2 at 37.degree. C. The medium with DNA is then removed
from the cells and the cells are further held in culture with
DMEM.
[0104] 2.4. Growth and Isolation of Individual Cell Colonies
[0105] The following day the transfected cells are diluted to a
concentration of 250,000 cells per culture bottle of 75 cm.sup.2
and these are further held in culture through selection with the
antibiotic G418. After 10-12 days individual colonies can be picked
up out of the culture bottle.
[0106] The selected colonies are cultured and analyzed for
expression of the L-galactono-.gamma.-lactone dehydrogenase. The
clones designated with V3, V6, V8 and V14 were found to be positive
for the expression of the L-galactono-.gamma.-lactone
dehydrogenase. The enzyme activities are shown in table 6.
4TABLE 6 GLDase activity in transfected murine fibrosarcoma cells.
All values are expressed in specific activity of the enzyme
(units/min./mg protein). cell line: VI pod (control) 0 N2 (control)
0 transfected: V6 3.7 V14 2.1 V3 1.4 V8 2.0
[0107]
5TABLE 1 Purification diagram for GLDase A mitochondrial extract of
15 kg cauliflower florets was used for the preparation. TOTAL
SPECIFIC VOL. PROTEIN ACTIVITY ACTIVITY STEP (ml) (mg) units
units/mg -FOLD YIELD Acetone prec. 2500 1510 44,900 30.5 1 100 DEAE
ion 83 54.7 46,500 845 28 104 exchanger Phenyl Sepha- 38 21.2
30,800 1,467 49 69 rose Gel fil- 54 10.5 20,900 1,900 63 47 tration
FPLC Resource 32 0.3 8,100 2,700 900 18 Q FPLC Poros 20 4 0.01 508
50,800 1693 1.1 SP
[0108]
6TABLE 2 Amino acid sequences determined from the GLDase
polypeptide Peptide sequences obtained from 55 kDa polypeptide
after tryptic digestion NH.sub.2-terminal sequences YAPLEXEDL
Internal sequences LXDQYSAYE (1) VNQAEAEF (2) LIALDPLNDVHVG (3)
YTTEEALK (4) WTGR (5) GTIELSK (6) VNQAEAEFWK (7) IEIPK (8) Peptide
sequences obtained from 31 kDa and 26 kDa subdivisions.
NH.sub.2-terminal sequences APLPDLHTVSN (30 kDa) XSSKKTPDXRXPDINXL
(26 kDa) X refers to amino acid sequences not determined by
sequence determining runs. Degenerated oligonucleotides were
designed on the basis of peptides 1, 4 and 7.
* * * * *