U.S. patent application number 10/615144 was filed with the patent office on 2004-09-16 for plant gntl sequences and the use thereof for the production of plants having reduced or lacking n-acetyl glucosaminyl transferase i (gnti) activity.
Invention is credited to Schaewen, Antje Von.
Application Number | 20040181827 10/615144 |
Document ID | / |
Family ID | 7851259 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040181827 |
Kind Code |
A1 |
Schaewen, Antje Von |
September 16, 2004 |
Plant Gntl sequences and the use thereof for the production of
plants having reduced or lacking N-acetyl glucosaminyl transferase
I (GnTI) activity
Abstract
This invention relates to plant GntI sequences, in particular to
plant nucleic acid sequences encoding the enzyme N-acetyl
glucosaminyl transferase I (GnTI), DNA sequences derived therefrom,
including GntI antisense and sense constructs, and the translation
products thereof, antibodies directed against said translation
products, as well as the use of the sequence information for the
production of transformed microorganisms and transgenic plants,
including those having reduced or missing N-acetyl glucosaminyl
transferase I activity. Such plants displaying reduced or lacking
N-acetyl glucosaminyl transferase I activity are of great
importance for the production of glycoproteins of specific
constitution with respect to their sugar residues.
Inventors: |
Schaewen, Antje Von;
(Osnabruck, DE) |
Correspondence
Address: |
William H. Benz
Foley & Lardner LLP
Three Palo Alto Square
3000 El Camino Real, Suite 100
Palo Alto
CA
94306-2121
US
|
Family ID: |
7851259 |
Appl. No.: |
10/615144 |
Filed: |
July 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10615144 |
Jul 9, 2003 |
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09591466 |
Jun 9, 2000 |
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6653459 |
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09591466 |
Jun 9, 2000 |
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PCT/EP98/08001 |
Dec 9, 1998 |
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Current U.S.
Class: |
800/284 |
Current CPC
Class: |
C12N 15/8257 20130101;
C12N 9/1051 20130101; C12N 15/8242 20130101 |
Class at
Publication: |
800/284 |
International
Class: |
A01H 001/00; C12N
015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 1997 |
DE |
197 54 622.6 |
Claims
1. Method for the production of glycoproteins displaying minimal,
uniform and defined sugar residues, comprising cultivating a
transgenic plant, parts of transgenic plants or transformed plant
cells, and isolating the desired glycoprotein from the material
cultivated, characterized in that the transgenic plant, parts of
transgenic plants or transformed plant cells, respectively, is/are
transformed with an antisense construct or a sense construct,
comprising an antisense DNA or a sense DNA with respect to the DNA
sequence for a gene or a cDNA for plant N-acetyl glucosaminyl
transferase I or a part thereof, for elimination or reduction of
the activity of said N-acetyl glucosaminyl transferase, wherein the
antisense or sense construct optionally contains additional
regulatory sequences for the transcription of the respective
antisense or sense DNA.
2. Method according to claim 1, characterized in that for
transformation an antisense or sense construct with respect to one
of the cDNAs encoding N-acetyl glucosaminyl transferase I from
Solanum tuberosum, Nicotiana tabacum or Arabidopsis thaliana is
used.
3. Method according to claim 2, characterized in that for
transformation an antisense or sense construct with respect to one
of the DNA sequences given in SEQ ID NO: 1, 3 or 5 is used.
4. Method according to any of the claims 1 to 3, characterized in
that the transgenic plant used is additionally transformed with the
gene encoding the desired glycoprotein.
5. DNA, characterized in that it encodes N-acetyl glucosaminyl
transferase I from Solanum tuberosum.
6. DNA according to claim 5, characterized in that it comprises the
nucleotide sequence given in SEQ ID NO: 1 or a part thereof.
7. DNA, characterized in that it encodes N-acetyl glucosaminyl
transferase I from Nicotiana tabacum.
8. DNA according to claim 7, characterized in that it comprises the
nucleotide sequence given in SEQ ID NO: 3 or a part thereof.
9. DNA encoding N-acetyl glucosaminyl transferase I from
Arabidopsis thaliana, characterized in that said DNA encodes the
amino-acid sequence given in SEQ ID NO: 6 or the nucleotide
sequence given in SEQ ID NO: 5 or a part thereof.
10. DNA, characterized in that it comprises the nucleotide sequence
complementary to the DNA according to claim 6, 8 or 9.
11. DNA, characterized in that it may be obtained by substitution,
deletion and/or insertion of one or more nucleotides and/or
truncation at the 5' and/or 3' end of one of the DNAs according to
any of the claims 5 to 10, with the proviso, that said DNA
hybridizes at least in a partial region with the starting DNA or
its complementary sequence or parts thereof under stringent
conditions.
12. DNA, characterized in that it represents a gene or is part of a
gene, which encodes the enzyme N-acetyl glucosaminyl transferase I,
and which in its entirety or in a partial region thereof hybridizes
under stringent conditions to one of the DNA sequences or fragments
according to any of the claims 5 to 11 and/or to a DNA sequence,
which has been derived from the amino acid sequences given in SEQ
ID NO: 1, 3 and/or 5, considering the degeneration of the genetic
code.
13. DNA construct, characterized in that it comprises one or more
of the DNAs according to any of the claims 5 to 14.
14. DNA construct according to claim 13, characterized in that it
comprises an antisense or sense DNA with respect to the DNA
sequence according to any of the claims 5 to 12 and optionally
regulatory sequences for the transcription of the antisense or
sense DNA, respectively.
15. Vector, plasmid, cosmid, virus or phage genome, characterized
in that it contains at least a DNA and/or construct according to
any of the claims 5 to 14.
16. N-acetyl glucosaminyl transferase I from Solanum tuberosum.
17. N-acetyl glucosaminyl transferase I from Nicotiana tabacum.
18. N-acetyl glucosaminyl transferase I from Arabidopsis thaliana,
characterized in that the enzyme comprises the amino acid sequence
set forth in SEQ ID NO: 6.
19. N-acetyl glucosaminyl transferase I, characterized in that the
enzyme comprises the amino acid sequence set forth in SEQ ID NO:
2.
20. N-acetyl glucosaminyl transferase I, characterized in that the
enzyme comprises amino acids 74 to 446 of the amino acid sequence
set forth in SEQ ID NO: 2.
21. N-acetyl glucosaminyl transferase I, characterized in that the
enzyme comprises the amino acid sequence set forth in SEQ ID NO:
4.
22. N-acetyl glucosaminyl transferase I, available due to
hybridization of its gene or one or more of the portions of its
gene to one or more of the DNAs and/or DNA fragments according to
any of the claims 5 to 12.
23. Enzymes or proteins derived from the enzymes according to any
of the claims 16 to 22 by substitution, deletion, insertion and/or
modification of individual amino acids and/or smaller groups of
amino acids and/or by N- and/or C-terminal truncation and/or
extension.
24. Protein or peptide, comprising one or more portions of the
amino acid sequence(s) of one or more of the enzymes defined in any
of the claims 16 to 23.
25. Protein or peptide, encoded by one of the DNAs according to any
of the claims 5 to 12.
26. Antigen, characterized in that it comprises: the amino acid
sequence given in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, or
amino acids 74 to 446 of the amino acid sequence given in FIG. 2,
or an amino acid sequence derived from the amino acid sequences
given in SEQ ID NO: 2, 4 or 6 by substitution, deletion, insertion
and/or modification of individual amino acids and/or smaller groups
of amino acids, or one or more parts of said sequences, with the
proviso, that upon immunization of a host with the antigen, said
antigen may raise an immunological reaction, including the
production of antibodies directed against the antigen.
27. Monoclonal or polyclonal antibody, characterized in that it
specifically recognizes and binds one or more of the enzymes or
antigens according to any of the claims 16 to 26.
28. Microorganism, characterized in that it is transformed by at
least one of the nucleotide sequences selected from the DNAs,
constructs, vectors, plasmids, cosmids, virus or phage genomes
according to one or more of the claims 5 to 15.
29. Transgenic plant, transgenic seed, transgenic reproduction
material, parts of transgenic plants or transformed plant cell,
obtainable by integration of one or more DNA sequence(s) or
construct(s) according to any of the claims 5 to 13 under the
control of a promoter effective in plants, into the genome of a
plant, or via infection by means of a virus containing one or more
DNA sequence(s) or construct(s) according to any of the claims 5 to
13, for an extrachromosomal propagation and expression of the DNA
sequence(s) or construct(s) in the plant tissue infected.
30. Transgenic plant, transgenic seed, transgenic reproduction
material, parts of transgenic plants or transformed plant cell with
missing or reduced N-acetyl glucosaminyl transferase I activity,
obtainable by integration of one or more antisense or sense
construct(s) according to claim 14 under the control of a promoter
effective in plants, into the genome of a plant, or by viral
infection by means of a virus containing one or more antisense or
sense construct(s) according to claim 14, for an extrachromosomal
propagation and transcription of the antisense construct(s) in the
plant tissue infected.
Description
[0001] The present invention relates to plant GnTI sequences, in
particular, plant nucleic acid sequences encoding the enzyme
N-acetyl glucosaminyl transferase I (GnTI), as well as GntI
antisense or sense constructs, deduced therefrom, and their
translation products, antibodies directed against said translation
products as well as the use of the sequence information for the
production of transformed microorganisms and of transgenic plants,
including those with reduced or lacking N-acetyl glucosaminyl
transferase I activity. Such plants with reduced or lacking
N-acetyl glucosaminyl transferase I activity are of great
importance for the production of glycoproteins of specific
constitution with respect to their sugar residues.
PRIOR ART
[0002] In eukaryotes, glycoproteins are cotranslationally assembled
in the endoplasmatic reticulum (ER) (i.e. during import into the ER
lumen) by the attachment of initially membrane bound glycans (via
dolichol pyrophosphate) to specific asparagine residues in the
growing polypeptide chain (N-glycosylation). In higher organisms,
sugar units located at the surface of the folded polypeptide chain
are subjected to further trimming and modification reactions (ref.
1) in the Golgi cisternae. Initially, typical basic
Glc.sub.3Man.sub.9GlcNAc.sub.2 units of the high-mannose type are
formed by means of different glycosidases and glycosyl transferases
in the ER, which during the passage through the different Golgi
cisternae are subsequently converted to so-called complex glycans.
The latter are characterized by less mannose units and the presence
of additional sugar residues, such as fucose, galactose and/or
xylose in plants and sialic acid (N-acetyl neuraminic acid, NeuNAc)
in mammals (ref. 1,2,3). The extent of the modifications can differ
between glycoproteins. Single polypetide chains may carry
heterogeneous sugar chains. Furthermore, the glycosylation pattern
may vary for a specific polypeptide (tissue specific differences),
and does not always have to be uniform with respect to a specific
glycosylation site, which is referred to as microheterogeneity
(ref. 4,5). Up to now, the role of asparagine bound glycans is
barely understood, which i.a. results from the fact, that said
glycans may serve several functions (ref. 6). However, it can be
assumed, that e.g. protection of a polypeptide chain from
proteolytic degradation can also be achieved by glycans of a more
simple oligomannosyl type (ref.7).
DESCRIPTION OF PROBLEMS
[0003] Glycoproteins are highly important in medicine and research.
However, large scale isolation of glycoproteins is time-consuming
and expensive. The direct use of glycoproteins isolated
conventionally often raises problems, since upon administration as
a therapeutic, single residues of the glycan components may cause
undesired side effects. In this context, the glycan component
predominantly contributes to the physico-chemical properties (such
as folding, stability and solubility) of the glycoproteins.
Furthermore, isolated glycoproteins, as already mentioned above,
rarely carry uniform sugar residues, which is referred to as
microheterogeneity.
[0004] For the production of glycoproteins for medicine and
research, yeasts prove to be unsuitable, since they are only able
to perform glycosylations of the so-called high-mannose type. While
insects and higher plants exhibit complex glycoprotein
modifications, these, however, differ from those of animals.
Therefore, glycoproteins isolated from plants have a strong
antigenic effect in mammals. In most cases, animal organisms with
glycosylation defects are not viable, since terminal glycan
residues (e.g. of membraneous glycoproteins) mostly possess
biological signal function and are indispensable for
cell-cell-recognition during the course of embryogenesis. Mammalian
cell lines with defined glycosylation defects already exist, the
cultivation of which, however, is labour-intensive and
expensive.
[0005] For mammals, different glycosylation mutants have been
described in detail at the cell culture level (ref. 7,8,9,10). Said
mutants are either defective in biosynthesis of mature
oligosaccharide chains attached to dolichol pyrophosphate or in
glycan processing or show alterations in their terminal sugar
residues, respectively. Some of these cell lines exhibit a
conditional-lethal phenotype or are defective in intracellular
protein transport. The consequences of said defects for the intact
organism are difficult to estimate. It has been observed, that a
modification in the pattern of complex glycans on the cell surfaces
of mammals is accompanied by the formation of tumours and
metastases, although a functional relationship could not yet
unambiguously be demonstrated (ref. 9). Therefore, in mammals
glycosylation mutants are very rare. These defects, summarized
under HEMPAS (Hereditary Erythroblastic Multinuclearity with a
Positive Acidified Serum lysis test) (ref. 10,11), are based either
on a deficiency in mannosidase II and/or low levels of the enzyme
N-acetyl glucosaminyl transferase II (GnTII), and have strongly
limiting effects on the viability of the mutated organism. GntI
knock-out mice, in which the gene for GnTI has been destroyed,
already die in utero of multiple developmental defects (personal
communication, H. Schachter, Toronto).
[0006] Until recently, no comparable mutants were known for plants.
By the-use of- an antiserum, which specifically recognizes complex
modified glycan chains of plant glycoproteins and which
predominantly is directed against the highly antigenic
.beta.1.fwdarw.2 linked xylose residues (ref. 12), the applicant
was able to isolate several independant mutants from an EMS
mutagenized F2 population of the genetic model plant Arabidopsis
thaliana, which no longer showed complex glycoprotein modification
(complex glycan, cgl mutants). After at least five backcrosses,
each followed by intermittent selfings (to screen for the recessive
defects), the glycoproteins were analyzed. These glycoproteins
mainly carried glycans of the Man.sub.5GlcNAc.sub.2 type,
indicating a defect in N-acetyl glucosaminyl transferase I (GnTI)
(ref. 8). Indeed, the Arabidopsis cgl mutants lacked GnTI activity
(ref. 13), which normally catalyzes the first reaction in the
synthetic pathway to complex modified sugar chains (ref. 1).
However, according to observations so far, the viability of the
mutated plants is not affected. In recent publications, plants are
suggested as a putative source for the production of
pharmaceutically relevant glycoproteins or vaccines (ref. 14,15).
There however, it was overlooked, that glycoproteins isolated from
plants may cause severe immune reactions in mammals, which up to
now obstructed the production of heterologous glycoproteins in
cultivated plants.
[0007] The applicant could demonstrate by way of example for the
Arabidopsis cgl mutant, that plants can manage without complex
modified glycoproteins to a great extent (ref. 13). Initially,
secretory proteins are normally glycosylated in the ER of the
mutant. In the Golgi apparatus of the cgl mutant, however, the
oligomannosyl chains linked to the polypetide backbone via
asparagine residues (N-glycosylation) then remain at the stage of
Man.sub.5GlcNAc.sub.2 residues, since N-acetyl glucosaminyl
transferase I (GnTI) activity is missing (FIG. 1). By this
biosynthesis block, the plant specific complex glycoprotein
modification and in particular the attachment of .alpha.1.fwdarw.3
fucose and .beta.1.fwdarw.2 xylose residues is prevented, whereby
the strong antigenic effect on the mammalian organism is absent.
However, Arabidopsis as a herb only has little utilizable biomass.
Therefore, for the large scale production of biotechnologically
relevant glycoproteins these cgl plants are less suitable. As an
alternative, cultivars, especially Solanaceae, such as potato,
tobacco, tomato or pepper and furthermore alfalfa, canola, beets,
soybean, lettuce, corn, rice and grain, with missing or highly
reduced GnTI activity, would be ideal for the production of
heterologous glycoproteins in plants. For this purpose, the methods
of homology-dependent gene silencing would be applicable (ref. 16,
17).
[0008] As FIG. 3 demonstrates, the homology of the first determined
plant GntI sequence from potato (Solanum tuberosum L., St) is
extraordinary low in comparison to the corresponding known
sequences of animal organisms (only 30-40% identity at the protein
level, cf. FIG. 3A). Therefore, by the use of heterologous GntI
gene sequences an efficient reduction of endogenous complex
glycoprotein modification in plants by means of antisense or sense
suppression, respectively, (ref. 21), probably cannot be
achieved.
[0009] Thus, in medicine and research there is still the need for a
cost-effective production in suitable organisms of recombinant
glycoproteins with a minimum of uniform, i.e. defined sugar
residues.
NATURE OF THE PRESENT INVENTION
[0010] Since the applicant for the first time has been able to
isolate and elucidate plant GntI cDNA sequences, it is now possible
i.a. to obtain and, in particular, to generate any plant having
reduced or missing GnTI activity, and to detect the corresponding
mutants, respectively, by means of reverse genetic approaches
following transposon (ref. 18) or T-DNA insertion (ref. 19),
respectively, so as to produce glycoproteins with low antigenic
potential in said mutants.
[0011] i) Enzymes
[0012] Generally, the present invention comprises different
N-acetyl glucosaminyl transferase I enzymes (EC 2.4.1.101) from
plants, e.g. potato (Solanum tuberosum L.), tobacco (Nicotiana
tabacum L.) and Arabidopsis thaliana (L.). In particular, the
present invention relates to enzymes, which exhibit or contain the
amino acid sequences given in FIGS. 2 and 3B as well as in the
accompanying sequence protocol.
[0013] Further, the invention comprises enzymes, which are derived
from amino acid sequences of the above mentioned enzymes by amino
acid substitution, deletion, insertion, modification or by
C-terminal and/or N-terminal truncation and/or extension, and
which--if showing enzymatic activity--exhibit a specificity
comparable to that of the starting enzyme,. i.e. N-acetyl
glucosaminyl transferase I activity, and optionally a comparable
activity.
[0014] In the present context, by the term "comparable activity" an
activity is understood, which is in the range of up to 100% above
or below that of the starting enzyme. Accordingly, also comprised
by the invention are derived enzymes or proteins with very low or
completely lacking enzymatic activity, which is detectable by means
of one or more of the tests mentioned as follows. The enzyme
activity is determined by a standard assay, which is performed with
microsomal fractions either under radioactive conditions, e.g.
using UDP-[6-.sup.3H]GlcNAc as a substrate (ref. 13) or
non-radioactive conditions (HPLC method; ref 20). Plant GnTI
activity can be detected on the subcellular level in Golgi
fractions (ref. 21). On account of low yields, however, it is
almost impossible to enrich the enzyme from plants.
[0015] Alternatively, an enzyme derived according to the present
invention, may optionally be defined as an enzyme, for which a DNA
sequence encoding the enzyme can be determined or derived, which
hybridizes to a DNA sequence encoding the starting enzyme or to a
complementary sequence under stringent conditions, as defined as
follows.
[0016] For example, an enzyme derived in such a manner represents
an isoform, which comprises the amino acids 74 to 446 of the amino
acid sequence illustrated in FIG. 2 and in SEQ ID No:1 and 2. This
isoform i.a. lacks the membrane anchor formed by amino acids 10 to
29. As a result, this enzyme isoform may be located in the plant
cytosol.
[0017] As examples for C- and/or N-terminally extended proteins,
fusion proteins can be mentioned, comprising in addition to an
amino acid sequence according to the invention a further protein,
which e.g. exhibits a different enzymatic activity or which may be
easily detected in another manner, such as by means of fluorescence
or phosphorescence or on account of a reactivity with specific
antibodies or by binding to suitable affinity matrices.
[0018] Furthermore, the invention comprises fragments of said
enzymes, which optionally no longer exhibit any enzymatic activity.
Generally, these fragments show an antigenic effect in a host
immunized with said fragments, and may accordingly be employed as
an antigen for the production of monoclonal or polyclonal
antibodies by immunization of a host with those fragments.
[0019] Moreover, this invention also relates to N-acetyl
glucosaminyl transferase I enzymes from other varieties and plant
species, which are obtainable on account of hybridization of their
genes or one or more regions of their genes:
[0020] to one or more of the DNA sequences and/or DNA fragments of
the present invention, as discussed below and/or
[0021] to suitable hybridization probes according to the invention,
which may be prepared on the basis of the amino acid sequences
mentioned in the sequence protocol considering the degeneration of
the genetic code.
[0022] Further comprised by the invention in accordance with the
above are enzymes or proteins derived from these N-acetyl
glucosaminyl transferase I enzymes, including fusion proteins
thereof, as well as fragments of all of these enzymes or
proteins.
[0023] ii) Antibodies
[0024] Another aspect of the present invention relates to the use
of the amino acid sequences mentioned above and of fragments
thereof having antigenic effects, respectively, for the production
of monoclonal or polyclonal antibodies or antisera by immunizing
hosts with said amino acid sequences or fragments, respectively, as
well as of antibodies or antisera, respectively, per se, which
specifically recognize and bind to the enzymes and/or antigens
described above. The general procedure and the corresponding
techniques for the generation of polyclonal and monoclonal
antibodies are all well-known to the persons skilled in the
art.
[0025] Exemplarily, by the use of a fragment of the GntI cDNA
(nucleotides 275 to 1395) represented in FIG. 2 and SEQ ID NO: 1,
the recombinant GnTI protein from Solanum tuberosum with 10
N-terminal histidine residues (His-tag) was overexpressed in E.
coli, and, following affinity purification via a metal-chelate
matrix, was employed as an antigen for the production of polyclonal
antisera in rabbits (cf. Examples 5 and 6).
[0026] One possible use of the antibodies of the invention resides
in the screening of plants for the presence of N-acetyl
glucosaminyl transferase I.
[0027] Binding of the antibody according to the present invention
to plant protein(s) indicates the presence of N-acetyl glucosaminyl
transferase I enzyme detectable with said antibody. In general,
this antibody may then be covalently bound to a carrier in a later
step, and optionally be employed for the enrichment or purification
of the enzyme by means of column chromatography.
[0028] On the other hand, a negative binding result using the
antibody of the present invention, i.e. lack of binding to the
plant proteins, may suggest, that N-acetyl glucosaminyl transferase
I enzyme is absent (or highly modified by mutation), and thus, that
N-acetyl glucosaminyl transferase I activity of a plant
investigated is missing or highly reduced.
[0029] Techniques for the realization of the screening assays
mentioned above or the enrichment or purification of enzymes by the
use of antibody columns or other affinity matrices (cf. Examples 5
and 6) are well-known to those skilled in the art.
[0030] iii) DNA Sequences
[0031] The present invention further comprises DNA sequences
encoding the amino acid sequences of the invention, including amino
acid sequences derived therefrom according to the above provisions.
In particular, the invention relates to the respective gene, which
is the basis of the amino acid sequences described in the FIGS. 2
and-3B and the sequence protocol, and especially, to the cDNA
sequences described in FIG. 2 and the sequence protocol, as well as
to DNA sequences derived from these genes and DNA sequences.
[0032] By the term "derived DNA sequences" are meant sequences,
which are obtained by substitution, deletion and/or insertion of
one or more and/or smaller groups of nucleotides of the sequences
mentioned above and/or by truncation or extension at the 5' and/or
3' terminus. Modifications within the DNA sequence may lead to
derived DNA sequences, which encode amino acid sequences being
identical to the amino acid sequence encoded by the starting DNA
sequence, or to such sequences, in which, compared to the amino
acid sequence, which is encoded by the starting DNA sequence,
single or a few amino acids are altered, i.e. substituted, deleted
and/or inserted, as well as to such sequences, which--optionally in
addition--are truncated and/or extended at the C-terminus and/or
N-terminus.
[0033] Furthermore, the present invention also extends to the
complementary sequences of the genes and DNA sequences according to
the invention, as well as the RNA transcription products
thereof.
[0034] Particularly comprised by the present invention are all
sequences derived according to the above provisions, which over
their entire length or only with one or more partial regions
hybridize under stringent conditions to the starting sequences
mentioned above or to the sequences complementary thereto or to
parts thereof, as well as DNA sequences comprising such
sequences.
[0035] By the term "hybridization under stringent conditions" in
the sense of the present invention is understood a hybridization
procedure according to one or more of the methods described below.
Hybridizing: up to 20 h in PEG buffer according to Church and
Gilbert (0.25 M Na.sub.2HPO.sub.4, 1 mM EDTA, 1% (w/v) BSA, 7%
(w/v) SDS, pH 7.5 with phosphoric acid; ref. 22) at 42.degree. C.
or in standard hybridization buffers with formamide at 42.degree.
C. or without formamide at 68.degree. C. (ref. 23). Washing: 3
times at 65.degree. C. for 30 min in 3.times.SSC buffer (ref. 23),
0.1% SDS.
[0036] In the sense of the present application, the term
"hybridization" always means hybridization under stringent
conditions, as mentioned above, even if this is not explicitely
indicated in the individual case.
[0037] Moreover, the invention relates to fragments of the DNA
sequences mentioned above, including the DNA sequences derived in
accordance with the above provisions, to fragments derived from
such fragments by nucleic acid substitution, insertion and/or
deletion as well as the corresponding fragments with sequences
complementary thereto. Such fragments are i.a. suitable as
sequencing or PCR primers, screening probes and/or for uses as
discussed below. For the use as a screening or hybridizing probe,
the DNA fragments according to the present invention are frequently
employed as radio-labelled fragments. Fragments carrying sequences,
which are derived from the starting sequences defined above by
substitution, deletion and/or insertion of one or more nucleotides,
and the sequences complementary thereto, respectively, are
comprised by the invention to that extent, as said fragments
hybridize under the above mentioned stringent conditions to the
starting sequences, or to the sequences complementary thereto,
respectively.
[0038] On the basis of the DNA sequences mentioned in the sequence
protocol and in FIG. 2, DNA fragments according to the invention
may for example be obtained starting from plant DNA by means of
restriction endonucleases using appropriate restriction sites or by
employment of PCR by means of primers appropriately synthesized, or
may, as an alternative, also be chemically synthesized. Such
techniques are well-known to those skilled in the art.
[0039] Moreover, the invention relates to any DNA sequences, which
represent a gene or are a part of a gene encoding the enzyme
N-acetyl glucosaminyl transferase I and, which in their entirety or
in a partial region thereof hybridize under stringent
conditions
[0040] to one or more of the DNA sequences of the invention
and/or
[0041] to one or more of the DNA fragments of the invention
and/or
[0042] to a DNA sequence, which is derived from the amino acid
sequences mentioned in the sequence protocol considering the
degeneration of the genetic code.
[0043] For this purpose, hybridization or screening probes are used
as DNA fragments, which generally comprise at least 15 nucleotides,
typically between 15 and 30 nucleotides, and, if necessary,
substantially more nucleotides. As an example, the primers employed
in Example 1 may be used. Alternatively, DNA sequences of
appropriate length, derived from the DNA sequences mentioned in the
sequence protocol, may be used. As a third possibility, appropriate
hybridization probes according to the invention may be developed
starting from the amino acid sequences mentioned in the sequence
protocol considering the degeneration of the genetic code.
[0044] In this respect, a subject-matter of the present invention
are also genes encoding N-acetyl glucosaminyl transferase I, which
may be detected from other varieties or plant species on account of
the hybridization thereof to above mentioned hybridization probes,
as well as DNA sequences, DNA fragments and constructs, which are
derived therefrom in accordance with the above provisions.
[0045] The isolation of the corresponding gene and sequencing
thereof following detection by means of the hybridization probes of
the invention are well within the skills of a specialist in this
field, and are detailed by way of example with respect to N-acetyl
glucosaminyl transferase I from Solanum tuberosum and to the
corresponding enzymes from Nicotiana tabacum and Arabidopsis
thaliana (partial sequence) in the examples.
[0046] Finally, another subject matter of the present invention are
antisense sequences with respect to any of the above DNA
sequences.
[0047] iv) Constructs
[0048] Also comprised by the invention are constructs, which may
optionally comprise besides additional 5' and/or 3' sequences, e.g.
linkers and/or regulatory DNA sequences or other modifications, the
DNA sequences of the invention, including the DNA sequences derived
as detailed above.
[0049] An example for this are hybridization or screening probes,
which in addition to a DNA sequence of the invention also comprise
a detection agent for the verification of hybridization products,
which in this case typically is non-radioactive, e.g. fluorescent
or phosphorescent molecules, biotin, biotin derivatives,
digoxigenin and digoxigenin derivatives. In this respect, however,
radioactive or non-radioactive detection agents may be considered,
which may be attached to the DNA sequence according to the present
invention e.g. by means of end labelling.
[0050] A subject-matter of the invention are also antisense and
sense constructs with respect to the DNA sequences and fragments
according to the present invention, i.e. with respect to
[0051] the DNA sequences mentioned in the sequence protocol and the
corresponding genes;
[0052] the DNA sequences derived therefrom in accordance with the
above provisions;
[0053] one or more regions of these DNA sequences;
[0054] DNA sequences, especially from other varieties or plant
species, which represent a gene or are a part of a gene, encoding
the enzyme N-acetyl glucosaminyl transferase I; and which hybridize
under stringent conditions
[0055] to one or more of the above DNA sequences and/or
[0056] to one or more of the above DNA fragments and/or
[0057] to a DNA sequence, which is derived from the amino acid
sequences mentioned in the sequence protocol considering the
degeneration of the genetic code.
[0058] Furthermore, the present invention extends to any
DNA-transfer systems such as vectors, plasmids, viral and phage
genomes or cosmids, which contain the DNA sequences according to
the present invention, e.g. the GntI gene, cDNA and DNA regions
according to the invention, as mentioned in the sequence protocol,
fragments thereof, in particular antisense or sense constructs
and/or cDNA sequences derived therefrom according to the above
provisions.
[0059] Various techniques for the production or synthesis of DNA,
DNA fragments, constructs and transfer systems according to the
invention, e.g. digestion by means of restriction endonucleases,
PCR amplification using suitable primers, optionally followed by
cloning and additional chemical or enzymatic modification starting
from plant DNA are well-known to those skilled in the art.
[0060] One possibility of application of the DNA hybridization
probes according to the invention is the detection of N-acetyl
glucosaminyl transferase I genes in plants other than those, from
which the DNA sequences mentioned in the sequence protocol were
obtained, or the detection of potential (other) isoforms of the
N-acetyl glucosaminyl transferase I gene in the starting plants
Solanum tuberosum, Nicotiana tabacum and Arabidopsis thaliana.
[0061] If it is possible to make use of a plant genomic library or
cDNA library for the hybridization experiment, a positive
hybridization result of such screening of each library may indicate
a clone or a few clones, which contain the desired sequence
completely or in part, i.e. the N-acetyl glucosaminyl transferase I
gene, combined with only a limited amount of other DNA from the
genome of the target plant, which appropriately facilitates cloning
and sequencing of the target gene. As an alternative, a PCR
amplification of the gene or parts thereof may also be carried out
starting from plant DNA and suitable constructs, so-called PCR
primers, to facilitate cloning and sequencing.
[0062] One use of sequencing primers of the invention, which are
synthesized starting from suitable regions of the sequences
according to the invention, e.g. enables genomic sequencing
starting from the entire target plant-genomic DNA cleaved by
restriction endonucleases, by means of the Church-Gilbert
technique, as well as sequencing at the cDNA level following RT-PCR
amplification of the total RNA of the target plant (cf. Expl.
1).
[0063] An alternative possibility of application of the DNA
hybridization probes according to the present invention derived
from the DNA sequences mentioned in the sequence protocol, is the
use thereof according to the invention for the detection of plants
with reduced or lacking N-acetyl glucosaminyl transferase I
activity. The hybridization experiment serves to detect the
N-acetyl glucosaminyl transferase I (GntI) gene by which it may be
concluded, e.g. owing to a negative hybridization result under
stringent conditions, that the GntI gene, and thus, N-acetyl
glucosaminyl transferase I activity in a plant investigated is
lacking.
[0064] Such hybridization techniques for the detection of proteins
or genes particularly in plant material by means of DNA probes are
also known to the persons skilled in the art. In this context, it
is referred to the above statements under item iii) for possible
hybridization conditions. Generally, suitable DNA hybridization
probes comprise at least 15 nucleotides of a sequence, which for
example is derived from the cDNA sequences mentioned in FIG. 2 and
the sequence protocol or from the corresponding GntI genes.
[0065] v) Transformed Microorganisms
[0066] Furthermore, the invention relates to microorganisms, such
as bacteria, bacteriophages, viruses, unicellular eukaryotic
organisms, such as fungi, yeasts, protozoa, algae, and human,
animal and plant cells, which have been transformed by one or more
of the DNA sequences of the invention or one or more of the
constructs of the invention, as illustrated above.
[0067] Transformed microorganisms according to the present
invention are used e.g. as expression systems for the transforming
foreign DNA to obtain the corresponding expression products. For
this purpose, typical microorganisms are bacteria, e.g. such as E.
coli. Furthermore, transformed microorganisms according to the
invention, in particular agrobacteria, may be employed e.g. for the
transformation of plants by transmission of the transforming
foreign DNA.
[0068] Methods for the transformation of cells of microorganisms by
(foreign) DNA are well-known to those skilled in the art.
[0069] For this purpose, e.g. constructs referred to as expression
vectors are used, which contain the DNA sequence of the invention
under control of a constitutive or inducible promoter, which, if
necessary, is additionally tissue specific, so as to enable the
expression of the introduced DNA in the target or host cell.
[0070] Therefore, a further aspect of the invention is a method for
the production of the enzymes and proteins of the invention by
using one or more of the transformed microorganisms of the present
invention. The method comprises cultivating at least one
microorganism transformed by the DNA of the invention, in
particular by one of the cDNAs mentioned in the sequence protocol,
under the control of an active promoter, as defined above, and
isolating the enzyme of the invention from the microorganisms, and,
if applicable, also from the culture medium. It is understood, that
this method also relates to the production of enzymes and proteins,
respectively, which are derived from the enzymes according to the
present invention from Solanum tuberosum, Nicotiana tabacum and
Arabidopsis thaliana, as defined under i) above.
[0071] Methods for the cultivation of transformed microorganisms
are well-known to those skilled in the art. For example, the
isolation of the expressed enzyme may be employed according to the
method described in Example 5 by means of metal-chelate
chromatography or, alternatively, by chromatography via columns,
which contain the antibodies against the enzyme bound to the
packing material.
[0072] vi) Transgenic Plants
[0073] Furthermore, the invention comprises transgenic plants,
which are transformed by means of a DNA sequence according to the
invention or a corresponding construct, respectively. Accordingly,
there may be obtained e.g. transgenic plants, in which a GnTI
deficiency, for example on account of a missing or defectice GntI
gene or due to defects in the regulatory regions of this gene, has
been removed by complementation using a construct derived from the
cDNA sequences mentioned in the sequence protocol, wherein the
expression of said construct is under the control of an active
constitutive or inducible promoter, which may be additionally
tissue specific. In this case, the GnTI enzyme or protein expressed
on account of the DNA of the invention contained in the construct
and having GnTI activity complements the GnTI activity missing in
the starting plant.
[0074] Also considered are transgenic plants, in which the GnTI
activity already present in the starting plant is increased by
additional expression of the GntI transgene introduced by means of
a construct according to the present invention. Up to now, the
extremely low expression of the GntI gene in vivo accompanied by
extremely low enzyme activity, which correspondingly was very
difficult to detect, has been a main problem in the investigation
of the enzyme N-acetyl glucosaminyl transferase I in plants. The
problem of a too low GnTI enzyme activity in plants may be overcome
by the coexpression of a DNA according to the present
invention.
[0075] In this case, it may be preferable for the transformation of
plants to employ DNA according to the invention, additionally
comprising a sequence region, which following expression enables a
facilitated detection and/or enrichment and purification,
respectively, of the protein product having GnTI activity. This is
for example accomplished by the use of a specific DNA sequence for
the expression of a recombinant GnTI enzyme, said sequence carrying
a N-terminal or C-terminal sequence extension encoding an affinity
marker. If it is additionally intended to provide an amino acid
sequence portion between the GnTI enzyme and the affinity marker,
which represents a recognition site for a specific protease,
cleavage of the N-terminal or C-terminal sequence extension from
the GnTI enzyme may be achieved by the subsequent use of this
specific protease, and the GnTI enzyme thereby obtained in isolated
form.
[0076] An example for this is the use of a DNA sequence according
to the present invention, which codes for the recombinant GnTI
enzyme with a C-terminal sequence extension, encoding the affinity
marker AWRHPQFGG (strep-tag; ref. 39), and an intervening protease
recognition site IEGR. The expression of the DNA according to the
present invention provides GnTI enzymes with the C-terminal
sequence extension mentioned, by means of which the expressed
protein molecules specifically bind to a streptavidin derivatized
matrix, and may thus be isolated. Then, by means of the protease
factor Xa specifically recognizing the amino acid sequence IEGR,
the GnTI portion of the protein molecules may be released. As an
alternative, the complete protein may be removed from the
streptavidin derivatized matrix by means of biotin or biotin
derivatives.
[0077] A further example is represented by DNA sequences of the
invention, encoding a protein which comprises multiple, e.g. 10,
N-terminally added histidine residues (His-tag) in addition to a
GnTI enzyme. Due to the N-terminal histidine residues, isolation or
purification, respectively, of the proteins expressed may be easily
conducted by metal-chelate affinity chromatography (e.g. Ni
sepharose) (cf. also Example 5).
[0078] Moreover, the invention comprises portions of such
transgenic plants, adequately transformed plant cells, transgenic
seeds and transgenic reproduction material.
[0079] A further important aspect of the invention is the use of
the sequence information discussed above for the production of
plants having reduced or lacking N-acetyl glucosaminyl transferase
I activity.
[0080] The possibilities of identifying plants with reduced or
lacking N-acetyl glucosaminyl transferase I activity due to a gene
defect or a missing gene by means of antibodies of the invention or
screening or hybridization probes of the invention have already
been described above.
[0081] Two additional possibilities reside in the use according to
the invention of antisense or sense constructs, respectively, which
are derived from the DNA sequence of a plant GntI gene, for the
production of transgenic plants with reduced or lacking N-acetyl
glucosaminyl transferase I activity by means of homology-dependent
gene silencing (cf. ref. 16,17). The DNA sequence used as a
starting sequence for the generation of the constructs, may be
derived from the starting plant to be transformed itself but also
from a different plant variety or species. In particular, antisense
or sense constructs as discussed under items iii) and iv) above are
of use. Generally, the constructs employed comprise at least 50 to
200 and more base pairs.
[0082] In particular, the constructs employed for this purpose
comprise at least 50 to 200 and more base pairs, with a sequence,
which is derived on the basis of
[0083] the cDNA sequences mentioned in the sequence protocol and/or
the corresponding GntI genes and/or
[0084] the derived DNA sequences discussed above and/or DNA
fragments according to the present invention and/or
[0085] the DNA sequences, in particular from other varieties and
plant species,.which encode N-acetyl glucosaminyl transferase I and
which may be identified due to a hybridization under stringent
conditions to hybridization or screening probes, as defined under
items iii) and iv) above.
[0086] Generally, the constructs contain a strong constitutive or
inducible promoter, which additionally may be tissue specific, by
means of which the antisense or sense DNA sequence regions are
controlled.
[0087] In the production of transgenic plants by integration of
antisense construct(s) into the plant genome or by viral infection
of starting plants or plant cells by means of virus containing
antisense construct(s) for an extrachomosomal propagation and
transcription of the antisense construct or the antisense
constructs in infected plant tissue, it is intended to achieve a
hybridization of GntI-gene transcripts to transcripts of the
antisense DNA region at the RNA level, which prevents translation
of the GntI mRNA. The result is a transgenic plant with strongly
decreased contents of N-acetyl glucosaminyl transferase I, and
thus, a strongly decreased corresponding enzyme activity.
[0088] For the transformation of plants according to the invention
with antisense constructs, for example constructs may be employed,
which hybridize to one of the complete cDNAs, mentioned in FIG. 2
and in the sequence protocol, or to corresponding regions thereof,
generally comprising at least 50 to more than 200 base-pairs.
Moreover, particularly preferred is the use of fragments, the
transcripts of which additionally cause a hybridization to a
portion of the 5' untranslated region of the GntI mRNA, at which or
in the proximity of which usually the attachment of ribosomes would
occur. Examples of such constructs are shown in FIG. 4.
[0089] In view of the occurence of an isoform in Solanum tuberosum,
which probably is located in the cytoplasm due to lack of the
membrane anchor (aa 10 to 29) of yet unkown function, it may be
desirable to target only the N-acetyl glucosaminyl transferase I
enzyme located in the Golgi cisternae, i.e. only that enzyme
comprising the membrane anchor. One reason for this desire may be
the effort or, in the individual case, also the requirement, to
affect as little as possible the cytoplasmatic metabolism of the
plant cell, for which the cytoplasmatic N-acetyl glucosaminyl
transferase I possibly is of importance. For this purpose,
antisense constructs may be used according to the present
invention, which themselves or the transcripts of which,
respectively, hybridize to a DNA or RNA region of the GntI gene or
the GntI mRNA, comprising a part of the 5' untranslated region and
the coding region including the membrane anchor. Generally, the
extension of the region of hybridization up to position 266 of the
cDNA in FIG. 2 and SEQ ID NO: 1 is considered harmless for the
purpose mentioned above.
[0090] In the production of transgenic plants by integration of
sense constructs into the plant genome or by viral infection of
starting plants or plant cells by means of virus containing sense
construct(s) for extrachromosomal propagation and expression of the
construct or constructs in infected plant tissue, there are assumed
hybridization phenomena in tobacco according to the work of Faske
et al. (ref. 17), of said constructs to the endogenous GntI gene at
a posttranscriptional or DNA level, respectively, which finally
affect or prevent the translation of the GntI gene. Also in this
case, the result are transgenic plants having reduced or-even
lacking N-acetyl glucosaminyl transferase I activity.
[0091] Methods for the stable integration of such antisense and
sense constructs into the genome of plants, or for the viral
infection of plants or plant cells, respectively, for an
extrachromosomal propagation and transcription/expression of such
constructs in infected plant tissue are well known to those skilled
in the art. This includes the direct DNA transfer (e.g. into
protoplasts by means of electroporation or by the addition of a
high molecular osmotic agent as well as biolistic methods, by which
DNA coated particles are shot into the plant tissue), such as the
use of natural host/vector systems (e.g. agrobacteria or plant
viruses). For viral infection of starting plants or plant cells by
viruses containing appropriate constructs for extrachromosomal
propagation and transcription/expression of the constructs in
infected plant tissue, a variety of specific viruses, such as
tobacco mosaic virus (TMV) or potato virus X, is available.
[0092] Representative plants, which are suitable for such
integration, comprise dicotyledonous as well as monocotyledonous
cultivated plants, in particular Solanaceae such as potato,
tobacco, tomato and pepper. Additionally, banana, alfalfa, canola,
beets, soybean, lettuce, corn, rice and grain, would be suitable
target plants for the use of homologous antisense constructs. For
example, the sequence from Arabidopsis thaliana mentioned in the
sequence protocol appears to be particularly suitable as a starting
sequence for the transformation according to the invention of
Brassicaceae, such as canola plants, by means of sense or antisense
constructs. Further plants of interest are any plants, which
express glycoproteins of interest for medicine and research.
[0093] Generally, it should be noted, that the transformation
according to the invention of plants, which in the corresponding
region of the GntI gene exhibit a homology of .gtoreq.70% at the
nucleotide level to the employed antisense or sense constructs
according to the present invention, typically results in transgenic
plants of the invention, which show the desired reduction of
N-acetyl glucosaminyl transferase I activity.
[0094] Further, another possibility is seen in the targeted
destruction (knock-out) of the N-acetyl glucosaminyl transferase I
gene via gene targeting by means of homologous recombination (ref.
24) in a target plant using a suitable DNA fragment derived from
the cDNA sequence of the present invention, similar to the
procedure established for yeast systems and mammals.
[0095] Further, the present invention comprises transgenic plants,
which have been transformed by the antisense or sense constructs
mentioned above or the viruses containing the same, respectively,
as well as parts of such transgenic plants, correspondingly
transformed plant cells, transgenic seeds and transgenic
reproduction material.
[0096] Methods of the production of transgenic plants, e.g. by
means of agrobacteria- or virus-mediated as well as direct DNA
transfer are known to those skilled in the art. Concerning
representative plants for such a transformation, the above
mentioned applies.
[0097] The plants of the invention and the plants obtained
according to the invention, respectively, with reduced or lacking
N-acetyl glucosaminyl transferase I activity, may be used according
to the invention for the production of glycoproteins with minimal
and uniform, i.e. defined, sugar residues. As discussed above, such
glycoproteins are of great importance for medicine and research. As
a reasonable source of raw material and food as well as due to
their unproblematical disposal via composting, plants per se
represent ideal bioreactors. According to the present invention, it
is now possible to express biotechnologically or pharmaceutically
relevant glycoproteins (e.g. therapeutics of low antigenic
potential for mammals) in cultivated plants, in which GnTI activity
is highly reduced or completely absent.
[0098] Accordingly, the invention also comprises a method for the
production of glycoproteins with minimal uniform and defined sugar
residues, comprising cultivating a transgenic plant according to
the invention, of parts of such plants or of plant cells
transformed according to the invention, each expressing the desired
glycoprotein, as well as isolating the desired glycoprotein from
the cultivated material.
[0099] In this context, representative cultivated plants are
Solanaceae, in particular potato, tobacco, tomato and pepper.
Furthermore possible are banana, alfalfa, canola, beets, soybean,
lettuce, corn, rice and grain.
[0100] The sequence of the enzymatically controlled and plant
specific N-glycan modifications, which secretory glycoproteins are
subjected to during passage through the Golgi apparatus of higher
plants, is schematically shown in FIG. 1. The biosynthesis block
due to lacking or insufficient N-acetyl glucosaminyl transferase I
(GlcNAc transferase I) activity in a plant leads, instead of
complex glycans, to the predominant formation of glycans of the
Man.sub.5GlcNAc.sub.2 type, i.e. glycoproteins with uniform and
well-defined sugar residues, which are of extremely high importance
for medicine and research.
[0101] For this purpose, the genes encoding the desired
glycoproteins may be expressed in their natural producing plants,
which have been transformed according to the present invention e.g.
by means of antisense or sense constructs to yield transgenic
plants with reduced or missing N-acetyl glucosaminyl transferase I
activity.
[0102] There is also the possibility to use transgenic plants of
the invention displaying reduced or lacking N-acetyl glucosaminyl
transferase I activity, which additionally have been transformed by
the gene encoding the desired glycoprotein. In order to achieve
this, constructs may be employed, which contain the gene encoding
the desired glycoprotein under the control of a strong constitutive
or inducible promoter, which is optionally tissue specific as well,
and lead to the integration of the gene into the plant genome.
Alternatively, the transformation may also be conducted by viral
infection by means of a virus containing the gene for the desired
glycoprotein for extrachromosomal propagation and expression of the
gene. The glycoprotein may then be expressed in the respective host
plant and obtained therefrom.
[0103] Naturally, as an alternative, the procedure may be such,
that initially a transformation using an expression construct or
virus containing the DNA encoding the glycoprotein is performed,
and subsequently, another transformation with one or more of the
antisense or sense constructs of the invention or with one or more
viruses, containing the corresponding DNA, is performed. It is also
possible to perform a simultaneous transformation using both
constructs or using one virus containing the antisense or sense
construct as well as the gene encoding the desired glycoprotein
(piggyback version).
[0104] Within the scope of the present invention, there is also
considered a viral overinfection of the transgenic plants according
to the invention, in which integration of an anti-sense/sense
construct and/or the gene encoding the desired glycoprotein into
the genome has already occured, by viruses containing the
antisense/sense construct and/or the gene encoding the desired
glycoprotein, for an additional extrachromosomal propagation and
transcription or expression, respectively, of this DNA. As a
result, the concentrations of antisense or sense DNA, respectively,
or of the expressed glycoprotein may be increased in the transgenic
plant cells.
[0105] It may prove to be practical for the production according to
the invention of glycoproteins with defined glycosylation, to use
tissue specific promoters in such cases, where it is intended to
obtain the desired glycoproteins specifically only from certain
parts of a plant such as tubers or roots. Today, for a large
variety of plant tissues, tissue specific promoters are available,
which drive expression of foreign genes specifically only-in these
tissues. By way of example, tuber specific promoters such as
patatin class I (ref. 26) and proteinase inhibitor II promoters
(ref. 27) may be mentioned. Under certain conditions, both
promoters exhibit expression also in leaf tissue, i.e. they can be
induced by high metabolite contents (for example sucrose) and in
the case of the proteinase inhibitor II promoter also by mechanical
lesion or by spraying with abscisic or jasmonic acid,
respectively.
[0106] The use of tissue specific promoters may also be indicated
in cases, where the DNA sequence or the transcription products or
translation products thereof according to the invention,
respectively, which are employed for the transformation, turn out
to be detrimental to certain plant parts, e.g. due to a negative
influence on the metabolism of the corresponding plant cells.
[0107] As a representative target glycoprotein, human
glucocerebrosidase may be used for the therapy of the hereditary
Gaucher's disease (ref. 25). In order to obtain human
glucocerebrosidase (GC) with uniform and defined sugar residues,
e.g. plants of the present invention which are transformed by means
of-antisense DNA, may be transformed with the gene encoding human
glucocerebrosidase. For this purpose, the human glucocerebrosidase
cDNA sequence (ref. 38) is modified at the 3' terminus by means of
PCR using gene specific primers in a manner, that the recombinant
enzyme carries a C-terminal sequence extension encoding an affinity
marker (e.g. AWRHPQFGG, strep-tag; ref. 39) and, optionally, also a
protease recognition site (e.g. IEGR) between the GnTI enzyme
region and the affinity marker. The GC-cDNA sequence thus altered
is expressed in GntI antisense plants of the present invention by
using a strong and optionally tissue specific promoter (e.g. for
potato under the control of the tuber specific B33 patatin
promoter), so that the enzyme synthesized in these plants
exclusively carries well defined N-glycans. The affinity marker is
intended to facilitate-the enrichment of the recombinant enzyme
from the transgenic plants. In this case, the expressed protein
molecules (GC-strep molecules) bind to a streptavidin derivatized
matrix via the affinity marker sequence and can be released
therefrom by means of biotin or biotin derivatives. The removal
from the strepatavidin derivatized matrix may also be carried out
by means of catalytic amounts of a protease, which exhibits a
specificity for the protease recognition site located between the
GnTI enzyme region and the affinity marker. In this case, only the
GnTI enzyme region is released from the matrix. This could be
advantageous especially in that case, if the affinity marker
sequence has a detrimental effect on the GnTI activity.
[0108] Due to their terminal mannose residues, the
Man.sub.5GlcNAc.sub.2-g- lycans of the glucocerebrosidase obtained,
from the plants of the present invention will be recognized by
macrophages as an uptake signal, and can thus directly be employed
for the therapy of hereditary Gaucher's disease. Currently, a
therapy is only possible upon expensive isolation and
deglycosylation of native glucocerebrosidase (ref. 25).
[0109] Accordingly, the production of recombinant glycoproteins may
be highly facilitated by the use of plant GntI sequences compared
to conventional methods, e.g. the chemical deglycosylation of
purified glycoproteins, which is technically demanding (ref. 25),
or a difficult and expensive production in GnTI deficient animal
cell lines (ref. 7,10).
DESCRIPTION OF THE FIGURES
[0110] FIG. 1: Sequence of plant specific N-glycan modifications,
which secretory glycoproteins are subject to during passage through
the Golgi apparatus of higher plants (ref. 28). The biosythesis
block to complex modified glycans is based on a deficiency in GnTI
activity (which is either caused by a defective or missing GnTI
enzyme or by effective reduction of the GntI gene expression) and
is indicated by a cross. Meaning of the symbols: (F) fucose
residues, (X) xylose residues, dues, (.circle-solid.) GlcNAc
residues, (.quadrature.) mannose residues.
[0111] FIG. 2: Full length cDNA sequence of a plant GnTI from
potato (Solanum tuberosum L.) and amino acid sequence deduced
therefrom. By way of example, the complete cDNA of the membrane
anchor containing GntI isoform from potato leaf tissue (A1) is
illustrated. The EcoRI/NotI linkers at the 5' and 3' ends of the
cDNA are highlighted by bold letters, the binding sites of the
degenerate oligonucleotides used for obtaining the RT-PCR probe are
underlined. In contrast to already published animal GnTI sequences,
the protein sequence derived from the potato cDNA clones contains a
potential N-glycosylation site: Asn-X(without Pro)-Ser/Thr, which
is indicated by an asterisk. The region of the membrane anchor is
highlighted in italics (aa 10 to 29). The start of the isoform
(A8), which is potentially located in the cytosol, is indicated by
an arrow.
[0112] FIG. 3: A, Degree of identity or similarity, respectively,
of the amino acid sequence deduced from a complete GntI cDNA
sequence from potato (A1) in comparison to other GnTI sequences of
animal organisms, which have been selected from data bases.
Identical amino acid positions (in %) are printed in bold letters,
similar amino acid positions are given in brackets underneath.
Meaning of the abbreviations: Hu, human; Ra, rat; Mo, mouse; Ce,
Caenorhabditis elegans (round-worm); St, Solanum tuberosum (potato)
B, Comparison of the derived amino acid sequences of different
plant GntI-cDNA clones. A_Stb-A1, GnTI from potato leaf; B_Ntb-A9,
GnTI from tobacco leaf (A9); C_Atb-Full, GnTI from Arabidopsis
thaliana. Identical aa are highlighted in black, similar aa in
light grey.
[0113] FIG. 4: Cloning strategy of the GntI-antisense constructs
used. Following fill-in of the ends, a NotI linker was introduced
into the SalI restriction site of the polylinker region of the
plant expression vector pA35 (=pA35N) (ref. 29), and the complete
A1-GntI-cDNA was inserted into pA35N via NotI. The corresponding
antisense construct (=pA35N-Alas) was inserted into binary vector
pBin19 (ref. 30) via EcoRI and HindIII. Additionally, following PCR
amplification, a 5' fragment of the A1-GntI-cDNA comprising 270 bp
was cloned into pA35N via XbaI and NotI restriction sites in
antisense orientation (=pA35N-A1-short) and also inserted into
pBin19. Abbreviations; Numerals in brackets, positions of the
restriction sites in the A1-GntI-cDNA (in base pairs); pBSK,
cloning vector (Stratagene): pGEM3Z, cloning vector (Promega);
CaMVp35S, constitutive 35S promoter of cauliflower mosaic virus;
OCSpA, polyadenylation signal of octopin synthase; PNOS, promoter
of nopaline synthase; NEO, neomycin phosphotransferase (selection
marker, confers kanamycin resistance); NOSpA, polyadenylation
signal of nopaline synthase; LB/RB, left/right border of the T-DNA
of the binary vector; arrow, translation initiation (ATG); A8,
start ;of the GnTI isoform, which is potentially located in the
cytosol (7 aa substitutions in comparison to A1).
[0114] FIG. 5: Extent of suppression of complex glycoprotein
modification in transgenic potato plants transformed with the long
GntI antisense construct (cf. FIG. 4). A, Coomassie-stained SDS gel
from leaf extracts; B, Western-blot analysis (Ref. 13,33) of
parallel samples developed with a complex-glycan antiserum (Ref.
12,13). The lanes contain 30 .mu.g each of total protein: cgl
(Ara), Arabidopsis cgl mutant (Ref. 13); WT(Desi), wild-type
potato; the numerals refer to individual transgenic potato plants;
the arrows represent molecular weight standards of 66, 45, 36 and
29 kDa, respectively.
[0115] FIG. 6: Detection of specificity of the generated GnTI
antiserum following cell fractionation (Ref. 40) of tobacco callus
material. For Western-blot analysis (Ref. 13,33) 30 .mu.g of
protein were applied per lane. The antiserum was used in 1:1000
dilution. Lane 1, homogenate following separation of cellular
debris; lane 2, vesicle fraction following column chromatography;
lane 3, sucrose gradient fraction I (microsomes); lane 4, sucrose
gradient fraction II (plastids); lane 5, antigen used for
immunization (recombinant GnTI fusion protein); arrow, molecular
weight of about 49 kDa.
EXPLANATION OF THE ABBREVIATIONS USED IN THE TEXT
[0116] Aa, amino acid(s); bp, base pair(s); EMS, Ethyl methane
sulfonate (mutagenic agent); F2, second filial generation; Fuc,
fucose; Glc, glucose; GlcNAc, N-acetyl glucosamine; GnTI, N-acetyl
glucosaminyl transferase I (EC 2.4.1.101); GntI, gene for GnTI
(nuclear encoded); kDa, kilodalton; Man, mannose; PCR, polymerase
chain reaction; PAGE, polyacrylamide gel electrophoresis; ref.,
reference; RT-PCR, reverse transcription coupled polymerase chain
reaction; SDS, sodium dodecyl sulfate; var., variety; Xyl,
xylose.
[0117] In the following, the invention will be described in more
detail by means of examples, which are only intended to illustrate
the invention and shall not limit the invention in any manner.
EXAMPLE 1
Isolation and Characterization of Plant GntI cDNA Clones
[0118] Total RNA was isolated from potato and tobacco leaf tissue,
and cDNA fragments of about 90 bp were amplified by means of RT-PCR
in combination with degenerate primers (procedure analogous to ref.
31), which were derived from conserved amino acid regions of known
GnTI sequences from animal organisms (sense primer 1*, 5'-TG(CT)
G(CT)I (AT)(GC)I GCI TGG (AC)A(CT) GA(CT) AA(CT)-3'; antisense
primer 3*, 5'-CCA ICC IT(AG) ICC (ACGT)G(CG) (AG)AA (AG)AA
(AG)TC-3'; 30 pmol of each primer per 50 .mu.l PCR assay at an
annealing temperature of 55.degree. C. and 45 cycles). Following
gel elution, the ends of the PCR products were repaired (i.e. blunt
ended using DNA polymerase I and phosphorylated using T4
polynucleotide kinase) and cloned into the EcoRV restriction site
of pBSK (Stratagene). By comparison with known GnTI sequences
between the primers (arrows), the identity of the derived amino
acid sequences from the potato and tobacco RT-PCR products could be
confirmed as being homologous; Q(R/M)QFVQDP(D/Y)ALYRS(homologous aa
are underlined). Of one clone each, radio-labelled probes were
synthesized by means of PCR (standard PCR assay using degenerate
primers as above, nucleotide mixture without dCTP, but instead with
50 .mu.Ci .alpha.-.sup.32P-dCTP [>3000 Ci/mMol]), and different
cDNA libraries were screened for GntI containing clones using the
corresponding homologous potato or tobacco probes, respectively
(procedure analogous to ref. 31; the stringent hybridization
conditions have already been described in the text above). The cDNA
libraries were prepared from mRNA of young and still growing plant
parts (sink tissues). Following cDNA synthesis and ligating
EcoRI/NotI adaptors (cDNA synthesis kit, Pharmacia) EcoRI
compatible lambda arms were ligated, those packaged and used to
transfect E. coli XL1 Blue cells (Lambda ZAPII cloning and
packaging system, Stratagene). Following amplification of the
libraries, one full-length GntI clone each was isolated from a
potato leaf sink library (Al according to FIG. 2 and SEQ ID NO: 1)
and a tobacco leaf sink library (A9 according to SEQ ID NO: 3), as
well as two additional clones from a tuber sink library (A6, A8).
The deduced GnTI amino-acid sequences contain a potential
N-glycosylation site, Asn-X(without Pro)-Ser/Thr, in contrast to
those of animals. One of the tuber GntI cDNA sequences carries stop
codons in all three reading frames in front of the first methionine
(A8). The coding region shows high homology to the longer tuber
clone (A6) (only 2 aa substitutions), but displays a completely
different 5' non-translated region. Furthermore, the membrane
anchor characteristic for the Golgi enzyme is missing, so that this
GnTI isoform might be located in the cytosol. Sequence comparisons
carried out by means of the gap or pileup option, respectively, and
the box option of the gcg software package (J. Devereux, P.
Haeberli, O. Smithies (1984) Nucl. Acids Res. 12: 387-395)
indicate, that the deduced plant GnTI amino-acid sequences exhibit
only 30-40% identity and 57-59% similarity to those of animal
organisms (FIG. 3A), while they are highly homologous among each
other (75 - 90% identity, FIG. 3B).
[0119] The procedure in the case of Arabidopsis thaliana was
analogous, wherein for the preparation of a specific probe first a
partial GntI sequence was amplified by RT-PCR using GntI sense
primer 4A (5'-ATCGGAAAGCTTGGATCC CCA GTG GC(AG) GCT GTA GTT GTT ATG
GCT TGC-3'; HindIII restriction site underlined, BamHI printed in
bold) and antisense primer 3*, as defined above. First, a
5'-incomplete cDNA clone was isolated from a phage library (Lambda
Uni-Zap) using this probe. By means of a vector insert PCR, the
missing 5'-terminus was amplified from another library (via an
unique SpeI restriction site in the 5' region) and assembled to
yield a full-length cDNA sequence. The nucleic acid sequence
determined by means of sequencing is listed in SEQ ID NO: 5.
EXAMPLE 2
Functional Complementation of a GnTI Defect Using GntI cDNA upon
Transient Expression in Protoplasts of the Arabidopsis Thaliana cgl
Mutant
[0120] Approximately 4 weeks subsequent to sowing, protoplasts were
isolated from leaves of cgl mutants cultivated under sterile
conditions (nonstainer plants following 5 backcrosses, ref. 13),
transformed with expression constructs of the complete GntI cDNA
sequences (NotI cDNA fragments, cf. FIG. 4) in sense (pA35N-A1s or
pA35N-A9s, respectively) or antisense orientation (pA35N-Alas or
pA35N-A9as, respectively), and cultivated for 96 h at room
temperature in the dark (50 .mu.g of plasmid DNA each per 1 million
protoplasts, PEG method according to ref. 32). Subsequent SDS-PAGE
of the protoplast extracts and Western-blot analysis (analogous to
ref. 13, 33) indicated functional complementation of the GnTI
defect, i.e. complex glycosylation of numerous protein bands upon
transient expression of the potato Al and tobacco A9 sense
constructs, but not of the corresponding antisense constructs in
protoplasts of the Arabidopsis cgl mutant (data not shown).
EXAMPLE 3
Cloning of the Binary Expression Constructs pBin-35-Alas and
pBin-35-A1-short (cf. FIG. 4)
[0121] Into the SalI restriction site of the polylinker region
(corresponding to the one of pUC18) of plant expression vector pA35
(ref. 29), a NotI linker was introduced subsequently to the fill-in
of the ends (=pA35N), and the complete A1-GntI-cDNA (nucleotides 9
to 1657; according to the cDNA in FIG. 2) was inserted into pA35N
via NotI (sense construct pA35N-A1s and antisense construct
pA35N-Alas, respectively). The expression cassettes of the sense
and antisense constructs, respectively, were isolated via the
terminal restriction sites (filled-in NcoI restriction site,
partial post digestion with HindIII) as a fragment of about 2410 bp
and inserted into the EcoRI (filled-in) and HindIII restriction
sites of the binary vector pBin19 (Ref. 30) (=pBin-35-A1s and
pBin-35-Alas, respectively). The EcoRI restriction site of the
vector is restored by fusion with the equally filled-in NcoI
restriction site of the fragment. By means of a standard PCR assay
(sense primer: KS sequencing primer (Stratagene) extended for PCR,
5'-GGC CCC CCC TCG AGG TCG ACG GTA TCG-3'; antisense primer:
5'-GGGCCTCTAGACTCGAG AGC (CT)AC TAC TCT TCC TTG CTG CTG GCT AAT CTT
G-3', XbaI restriction site underlined, XhoI restriction site in
italics), there was additionally amplified a 5'-fragment of the
GntI cDNA at an annealing temperature of 50.degree. C. (nucleotides
9 to 261, according to the cDNA in FIG. 2 and SEQ ID NO: 1). The
PCR product was digested with XbaI (within the antisense primer)
and NotI (within the 5'-linker of the cDNA), isolated as a fragment
of about 260 bp and cloned into pA35N (=pA35N-A1-short). The
expression cassette of the short antisense construct was also
inserted into pBin19 (=pBin-35-A1-short) as a EcoRI/HindIII
fragment (about 1020 bp).
EXAMPLE 4
Transformation of Agrobacteria by Means of the Binary GntI
Constructs and Regeneration of Transgenic Potato and Tobacco
Plants, Respectively, from Infected Leaf Discs
[0122] The binary antisense GntI constructs (pBin-35-Alas and
pBin-35-A1-short) were transformed into the Agrobacterium strain
GV2260 (ref. 34, 35). By way of example, sterile leaf discs of
potato plants var. Dsire and of tobacco plants var. Wisconsin 38
were infected with the recombinant agrobacterial lines (50 .mu.l of
a fresh overnight culture in 10 ml liquid 2MS medium: 2% sucrose in
Murashige & Skoog salt/vitamin standard mediums pH 5.6; small
pieces of leaf without midrip; co-cultivation for 2 days in the
dark in phytotrons). Subsequent to washing of the infected leaf
pieces in 2MS medium with 250 .mu.g/ml claforan, transgenic plants
were regenerated from said pieces in tissue culture under kanamycin
selection (potato protocol ref. 26; tobacco protocol ref. 36) and
analyzed for reduced GnTI activity (exemplary shown in FIG. 5 for
transgenic potato plants). As apparent from FIG. 5, antisense
suppression of complex glycoprotein modifiaction was successful in
transgenic potato plant #439. The determined reduction of complex
glycoprotein modification was stable in this transformant over the
entire investigation period of several months and has been verified
in three tests which were performed in an interval of about l month
each. For the respective transgenic tobacco plants, analogous
results were obtained.
EXAMPLE 5
Production of Recombinant Potato GnTI Protein (for the Production
of Antibodies)
[0123] Recombinant GnTI carrying 10 additional N-terminal histidine
residues (His-tag) was produced in E. coli by means of the pET
system (Novagen) and purified by metal-chelate affinity
chromatography. A cDNA fragment comprising nucleotides 275-1395 of
the potato GntI cDNA (corresp. to aa 75-446, FIG. 2 and SEQ ID NO:
1 and 2, respectively) was amplified by standard PCR (annealing
temperature of 50.degree. C., 30 cycles, ref. 31) (sense primer
GntI-5'fus: 5'-CATGGATCC CTC GAG AAG CGT CAG GAC CAG GAG TGC CGG
C-3'; antisense primer GntI-3' stop: 5'-ATCCCGGGATCCG CTA CGT ATC
TTC AAC TCC AAG TTG-3'; XhoI and BamHI restriction sites,
respectively, are underlined, stop codon in italics), and inserted
into vector pET16b (Novagen) (=pET-His-A1) via the restriction
sites of the synthetic primer (5'-XhoI-GntI-BamHI-3'). Following
propagation and analysis in E. coli XL1-Blue (Stratagene) the
construct was stored as a glycerol culture. Competent E. coli
BL21(DE3) pLysS cells (Novagen) were transformed with pET-His-A1
for overexpression. Addtition of IPTG
(Isopropyl-1-thio-.beta.-D-galactopyran- oside, at 0.5-2 mM) to a
BL21 culture in logarithmic growth phase, initially induces the
expression of T7 RNA polymerase (from the bacterial chromosome),
and thus, also the expression of the recombinant fusion protein
under control of the T7 promoter in pET vectors (Novagen). By means
of metal-chelate chromatography using TALON matrix (Clontech),
recombinant potato GnTI was purified from induced BL21:pET-His-A1
cells under denaturating conditions via its His-tag (manufacturer's
protocol, Novagen), and the preparation was verified with respect
to homogeneity by means of SDS-PAGE.
EXAMPLE 6
Raising of Polyclonal Antibodies in Rabbits
[0124] Recombinant potato GnTI (from Expl. 5) was used as an
antigen. Following the harvest of some milliliters of pre-immune
serum, the rabbits were subcutaneously injected with 300-500 .mu.g
of affinity-purified protein together with 25 .mu.g of GMDP
adjuvant (Gerbu) in intervals of three weeks. Subsequent to three
basis injections, the- animals were bled from the ear vein 12 to 14
days after the respective successive injection (boost), the serum
harvested (ref. 37) and tested for recognition of recombinant GnTI
by Western-blot analyses (dilution 1:200 to 1:2000). The antiserum
of the boosts resulting in the lowest background-to-signal ratio
were mixed with 0.04% sodium azide, aliquoted and kept at
+4.degree. C. or for long-term storage at -20.degree. C.,
respectively. As shown in FIG. 6, Western-blot analyses of tobacco
callus cells (BY-2 suspension culture) revealed a t specific GnTI
signal in enriched microsomal fractions, which indicates, that
antibodies raised against the recombinant protein specifially
recognize plant GnTI. The detection was carried out with enriched
microsomal fractions (ER and Golgi vesicles), since--due to low
amounts--it is not possible to detect GnTI protein in crude plant
extracts by means of the employed Western-blot method.
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Sequence CWU 1
1
14 1 1669 DNA Solanum tuberosum misc_feature (659)...(667) function
Asn codon in this context is a potential glycosylation site;
product N-glycosylation consensus sequence; phenotype N-glycans
modulate protein properties; 1 gaattcgcgg ccgcctgaga aaccctcgaa
ttcaatttcg catttggcag ag atg aga 58 Met Arg 1 ggg aac aag ttt tgc
ttt gat tta cgg tac ctt ctc gtc gtg gct gct 106 Gly Asn Lys Phe Cys
Phe Asp Leu Arg Tyr Leu Leu Val Val Ala Ala 5 10 15 ctc gcc ttc atc
tac ata cag atg cgg ctt ttc gcg aca cag tca gaa 154 Leu Ala Phe Ile
Tyr Ile Gln Met Arg Leu Phe Ala Thr Gln Ser Glu 20 25 30 tat gta
gac cgc ctt gct gct gca att gaa gca gaa aat cat tgt aca 202 Tyr Val
Asp Arg Leu Ala Ala Ala Ile Glu Ala Glu Asn His Cys Thr 35 40 45 50
agt cag acc aga ttg ctt att gac aag att agc cag cag caa gga aga 250
Ser Gln Thr Arg Leu Leu Ile Asp Lys Ile Ser Gln Gln Gln Gly Arg 55
60 65 gta gta gct ctt gaa gaa caa atg aag cat cag gac cag gag tgc
cgg 298 Val Val Ala Leu Glu Glu Gln Met Lys His Gln Asp Gln Glu Cys
Arg 70 75 80 caa tta agg gct ctt gtt cag gat ctt gaa agt aag ggc
ata aaa aag 346 Gln Leu Arg Ala Leu Val Gln Asp Leu Glu Ser Lys Gly
Ile Lys Lys 85 90 95 tta atc gga gat gtg cag atg cca gtg gca gct
gta gtt gtt atg gct 394 Leu Ile Gly Asp Val Gln Met Pro Val Ala Ala
Val Val Val Met Ala 100 105 110 tgc agt cgt act gac tac ctg gag agg
act att aaa tcc atc tta aaa 442 Cys Ser Arg Thr Asp Tyr Leu Glu Arg
Thr Ile Lys Ser Ile Leu Lys 115 120 125 130 tac caa aca tct gtt gca
tca aaa tat cct ctt ttc ata tcc cag gat 490 Tyr Gln Thr Ser Val Ala
Ser Lys Tyr Pro Leu Phe Ile Ser Gln Asp 135 140 145 gga tca aat cct
gat gta aga aag ctt gct ttg agc tat ggt cag ctg 538 Gly Ser Asn Pro
Asp Val Arg Lys Leu Ala Leu Ser Tyr Gly Gln Leu 150 155 160 acg tat
atg cag cac ttg gat tat gaa cct gtg cat act gaa aga cca 586 Thr Tyr
Met Gln His Leu Asp Tyr Glu Pro Val His Thr Glu Arg Pro 165 170 175
ggg gaa ctg gtt gca tac tac aag att gca cgt cat tac aag tgg gca 634
Gly Glu Leu Val Ala Tyr Tyr Lys Ile Ala Arg His Tyr Lys Trp Ala 180
185 190 ttg gat cag ctg ttt cac aag cat aat ttt agc cgt gtt atc ata
cta 682 Leu Asp Gln Leu Phe His Lys His Asn Phe Ser Arg Val Ile Ile
Leu 195 200 205 210 gaa gat gat atg gaa att gct gct gat ttt ttt gac
tat ttt gag gct 730 Glu Asp Asp Met Glu Ile Ala Ala Asp Phe Phe Asp
Tyr Phe Glu Ala 215 220 225 gga gct act ctt ctt gac aga gac aag tcg
att atg gct att tct tct 778 Gly Ala Thr Leu Leu Asp Arg Asp Lys Ser
Ile Met Ala Ile Ser Ser 230 235 240 tgg aat gac aat gga caa agg cag
ttc gtc caa gat cct gat gct ctt 826 Trp Asn Asp Asn Gly Gln Arg Gln
Phe Val Gln Asp Pro Asp Ala Leu 245 250 255 tac cgc tca gac ttt ttt
cct ggt ctt gga tgg atg ctt tca aaa tca 874 Tyr Arg Ser Asp Phe Phe
Pro Gly Leu Gly Trp Met Leu Ser Lys Ser 260 265 270 act tgg tcc gaa
cta tct cca aag tgg cca aag gct tac tgg gat gac 922 Thr Trp Ser Glu
Leu Ser Pro Lys Trp Pro Lys Ala Tyr Trp Asp Asp 275 280 285 290 tgg
cta agg ctg aaa gaa aat cac aga ggt cga caa ttt att cgc cca 970 Trp
Leu Arg Leu Lys Glu Asn His Arg Gly Arg Gln Phe Ile Arg Pro 295 300
305 gaa gtt tgc aga acg tac aat ttt ggt gag cat ggt tct agt ttg ggg
1018 Glu Val Cys Arg Thr Tyr Asn Phe Gly Glu His Gly Ser Ser Leu
Gly 310 315 320 cag ttt ttt aag cag tat ctt gag cca att aag cta aat
gat gtc cag 1066 Gln Phe Phe Lys Gln Tyr Leu Glu Pro Ile Lys Leu
Asn Asp Val Gln 325 330 335 gtt gat tgg aag tca atg gac cta agt tac
ctt ttg gag gac aac tat 1114 Val Asp Trp Lys Ser Met Asp Leu Ser
Tyr Leu Leu Glu Asp Asn Tyr 340 345 350 gtg aaa cac ttt ggc gac ttg
gtt aaa aag gct aag ccc atc cac gga 1162 Val Lys His Phe Gly Asp
Leu Val Lys Lys Ala Lys Pro Ile His Gly 355 360 365 370 gct gat gct
gtt ttg aaa gca ttt aac ata gat ggt gat gtg cgt att 1210 Ala Asp
Ala Val Leu Lys Ala Phe Asn Ile Asp Gly Asp Val Arg Ile 375 380 385
cag tac aga gac caa cta gac ttt gaa gat atc gct cga cag ttt ggc
1258 Gln Tyr Arg Asp Gln Leu Asp Phe Glu Asp Ile Ala Arg Gln Phe
Gly 390 395 400 att ttt gaa gaa tgg aag gat ggt gta cca cgg gca gca
tat aaa ggg 1306 Ile Phe Glu Glu Trp Lys Asp Gly Val Pro Arg Ala
Ala Tyr Lys Gly 405 410 415 ata gta gtt ttc cgg ttt caa aca tct aga
cgt gtg ttc ctt gtt tcc 1354 Ile Val Val Phe Arg Phe Gln Thr Ser
Arg Arg Val Phe Leu Val Ser 420 425 430 cct gat tct ctt cga caa ctt
gga gtt gaa gat act tag cgaagatatg 1403 Pro Asp Ser Leu Arg Gln Leu
Gly Val Glu Asp Thr * 435 440 445 attggagcct gagcaacaat ttagacttat
ttggtaggat acatttgaaa gagctgacac 1463 gaaaagtatg actaccagta
gctacatgca acattttaat gttaatggaa ggaacccact 1523 gcttattgtt
ggaatggatg aatcatcacc acatcctatt attcaagttt acaaacataa 1583
agaggaaatg ttgccctata aaaacaaatt ttttgtttct aagaaggaac gttacgatta
1643 tgagcaactt tggcggccgc gaattc 1669 2 446 PRT Solanum tuberosum
2 Met Arg Gly Asn Lys Phe Cys Phe Asp Leu Arg Tyr Leu Leu Val Val 1
5 10 15 Ala Ala Leu Ala Phe Ile Tyr Ile Gln Met Arg Leu Phe Ala Thr
Gln 20 25 30 Ser Glu Tyr Val Asp Arg Leu Ala Ala Ala Ile Glu Ala
Glu Asn His 35 40 45 Cys Thr Ser Gln Thr Arg Leu Leu Ile Asp Lys
Ile Ser Gln Gln Gln 50 55 60 Gly Arg Val Val Ala Leu Glu Glu Gln
Met Lys His Gln Asp Gln Glu 65 70 75 80 Cys Arg Gln Leu Arg Ala Leu
Val Gln Asp Leu Glu Ser Lys Gly Ile 85 90 95 Lys Lys Leu Ile Gly
Asp Val Gln Met Pro Val Ala Ala Val Val Val 100 105 110 Met Ala Cys
Ser Arg Thr Asp Tyr Leu Glu Arg Thr Ile Lys Ser Ile 115 120 125 Leu
Lys Tyr Gln Thr Ser Val Ala Ser Lys Tyr Pro Leu Phe Ile Ser 130 135
140 Gln Asp Gly Ser Asn Pro Asp Val Arg Lys Leu Ala Leu Ser Tyr Gly
145 150 155 160 Gln Leu Thr Tyr Met Gln His Leu Asp Tyr Glu Pro Val
His Thr Glu 165 170 175 Arg Pro Gly Glu Leu Val Ala Tyr Tyr Lys Ile
Ala Arg His Tyr Lys 180 185 190 Trp Ala Leu Asp Gln Leu Phe His Lys
His Asn Phe Ser Arg Val Ile 195 200 205 Ile Leu Glu Asp Asp Met Glu
Ile Ala Ala Asp Phe Phe Asp Tyr Phe 210 215 220 Glu Ala Gly Ala Thr
Leu Leu Asp Arg Asp Lys Ser Ile Met Ala Ile 225 230 235 240 Ser Ser
Trp Asn Asp Asn Gly Gln Arg Gln Phe Val Gln Asp Pro Asp 245 250 255
Ala Leu Tyr Arg Ser Asp Phe Phe Pro Gly Leu Gly Trp Met Leu Ser 260
265 270 Lys Ser Thr Trp Ser Glu Leu Ser Pro Lys Trp Pro Lys Ala Tyr
Trp 275 280 285 Asp Asp Trp Leu Arg Leu Lys Glu Asn His Arg Gly Arg
Gln Phe Ile 290 295 300 Arg Pro Glu Val Cys Arg Thr Tyr Asn Phe Gly
Glu His Gly Ser Ser 305 310 315 320 Leu Gly Gln Phe Phe Lys Gln Tyr
Leu Glu Pro Ile Lys Leu Asn Asp 325 330 335 Val Gln Val Asp Trp Lys
Ser Met Asp Leu Ser Tyr Leu Leu Glu Asp 340 345 350 Asn Tyr Val Lys
His Phe Gly Asp Leu Val Lys Lys Ala Lys Pro Ile 355 360 365 His Gly
Ala Asp Ala Val Leu Lys Ala Phe Asn Ile Asp Gly Asp Val 370 375 380
Arg Ile Gln Tyr Arg Asp Gln Leu Asp Phe Glu Asp Ile Ala Arg Gln 385
390 395 400 Phe Gly Ile Phe Glu Glu Trp Lys Asp Gly Val Pro Arg Ala
Ala Tyr 405 410 415 Lys Gly Ile Val Val Phe Arg Phe Gln Thr Ser Arg
Arg Val Phe Leu 420 425 430 Val Ser Pro Asp Ser Leu Arg Gln Leu Gly
Val Glu Asp Thr 435 440 445 3 1737 DNA Nicotiana tabacum
misc_feature (733)...(741) function Asn codon in this context is a
potential glycosylation site; product N-glycosylation consensus
sequence; phenotype N-glycans modulate protein properties; 3
gaattcgcgg ccgccattga cttgatccta actgaacagg caaagtaaat ccagcgatga
60 aacactcata actgaacact gagagactat tcgctttctc ctaaagcctt
caatcgaatt 120 cgcacg atg aga ggg aac aag ttt tgc tgt gat ttc cgg
tac ctc ctc 168 Met Arg Gly Asn Lys Phe Cys Cys Asp Phe Arg Tyr Leu
Leu 1 5 10 atc ttg gct gct gtc gcc ttc atc tac aca cag atg cgg ctt
ttt gcg 216 Ile Leu Ala Ala Val Ala Phe Ile Tyr Thr Gln Met Arg Leu
Phe Ala 15 20 25 30 aca cag tca gaa tat gca gat cgc ctt gct gct gca
att gaa gca gaa 264 Thr Gln Ser Glu Tyr Ala Asp Arg Leu Ala Ala Ala
Ile Glu Ala Glu 35 40 45 aat cat tgt aca agc cag acc aga ttg ctt
att gac cag att agc ctg 312 Asn His Cys Thr Ser Gln Thr Arg Leu Leu
Ile Asp Gln Ile Ser Leu 50 55 60 cag caa gga aga ata gtt gct ctt
gaa gaa caa atg aag cgt cag gac 360 Gln Gln Gly Arg Ile Val Ala Leu
Glu Glu Gln Met Lys Arg Gln Asp 65 70 75 cag gag tgc cga caa tta
agg gct ctt gtt cag gat ctt gaa agt aag 408 Gln Glu Cys Arg Gln Leu
Arg Ala Leu Val Gln Asp Leu Glu Ser Lys 80 85 90 ggc ata aaa aag
ttg atc gga aat gta cag atg cca gtg gct gct gta 456 Gly Ile Lys Lys
Leu Ile Gly Asn Val Gln Met Pro Val Ala Ala Val 95 100 105 110 gtt
gtt atg gct tgc aat cgg gct gat tac ctg gaa aag act att aaa 504 Val
Val Met Ala Cys Asn Arg Ala Asp Tyr Leu Glu Lys Thr Ile Lys 115 120
125 tcc atc tta aaa tac caa ata tct gtt gcg tca aaa tat cct ctt ttc
552 Ser Ile Leu Lys Tyr Gln Ile Ser Val Ala Ser Lys Tyr Pro Leu Phe
130 135 140 ata tcc cag gat gga tca cat cct gat gtc agg aag ctt gct
ttg agc 600 Ile Ser Gln Asp Gly Ser His Pro Asp Val Arg Lys Leu Ala
Leu Ser 145 150 155 tat gat cag ctg acg tat atg cag cac ttg gat ttt
gaa cct gtg cat 648 Tyr Asp Gln Leu Thr Tyr Met Gln His Leu Asp Phe
Glu Pro Val His 160 165 170 act gaa aga cca ggg gag ctg att gca tac
tac aaa att gca cgt cat 696 Thr Glu Arg Pro Gly Glu Leu Ile Ala Tyr
Tyr Lys Ile Ala Arg His 175 180 185 190 tac aag tgg gca ttg gat cag
ctg ttt tac aag cat aat ttt agc cgt 744 Tyr Lys Trp Ala Leu Asp Gln
Leu Phe Tyr Lys His Asn Phe Ser Arg 195 200 205 gtt atc ata cta gaa
gat gat atg gaa att gcc cct gat ttt ttt gac 792 Val Ile Ile Leu Glu
Asp Asp Met Glu Ile Ala Pro Asp Phe Phe Asp 210 215 220 ttt ttt gag
gct gga gct act ctt ctt gac aga gac aag tcg att atg 840 Phe Phe Glu
Ala Gly Ala Thr Leu Leu Asp Arg Asp Lys Ser Ile Met 225 230 235 gct
att tct tct tgg aat gac aat gga caa atg cag ttt gtc caa gat 888 Ala
Ile Ser Ser Trp Asn Asp Asn Gly Gln Met Gln Phe Val Gln Asp 240 245
250 cct tat gct ctt tac cgc tca gat ttt ttt ccc ggt ctt gga tgg atg
936 Pro Tyr Ala Leu Tyr Arg Ser Asp Phe Phe Pro Gly Leu Gly Trp Met
255 260 265 270 ctt tca aaa tct act tgg gac gaa tta tct cca aag tgg
cca aag gct 984 Leu Ser Lys Ser Thr Trp Asp Glu Leu Ser Pro Lys Trp
Pro Lys Ala 275 280 285 tac tgg gac gac tgg cta aga ctc aaa gag aat
cac aga ggt cga caa 1032 Tyr Trp Asp Asp Trp Leu Arg Leu Lys Glu
Asn His Arg Gly Arg Gln 290 295 300 ttt att cgc cca gaa gtt tgc aga
aca tat aat ttt ggt gag cat ggt 1080 Phe Ile Arg Pro Glu Val Cys
Arg Thr Tyr Asn Phe Gly Glu His Gly 305 310 315 tct agt ttg ggg cag
ttt ttc aag cag tat ctt gag cca att aaa cta 1128 Ser Ser Leu Gly
Gln Phe Phe Lys Gln Tyr Leu Glu Pro Ile Lys Leu 320 325 330 aat gat
gtc cag gtt gat tgg aag tca atg gac ctt agt tac ctt ttg 1176 Asn
Asp Val Gln Val Asp Trp Lys Ser Met Asp Leu Ser Tyr Leu Leu 335 340
345 350 gag gac aat tac gtg aaa cac ttt ggt gac ttg gtt aaa aag gct
aag 1224 Glu Asp Asn Tyr Val Lys His Phe Gly Asp Leu Val Lys Lys
Ala Lys 355 360 365 ccc atc cat gga gct gat gct gtc ttg aaa gca ttt
aac ata gat ggt 1272 Pro Ile His Gly Ala Asp Ala Val Leu Lys Ala
Phe Asn Ile Asp Gly 370 375 380 gat gtg cgt att cag tac aga gat caa
cta gac ttt gaa aat atc gca 1320 Asp Val Arg Ile Gln Tyr Arg Asp
Gln Leu Asp Phe Glu Asn Ile Ala 385 390 395 cgg caa ttt ggc att ttt
gaa gaa tgg aag gat ggt gta cca cgt gca 1368 Arg Gln Phe Gly Ile
Phe Glu Glu Trp Lys Asp Gly Val Pro Arg Ala 400 405 410 gca tat aaa
gga ata gta gtt ttc cgg tac caa acg tcc aga cgt gta 1416 Ala Tyr
Lys Gly Ile Val Val Phe Arg Tyr Gln Thr Ser Arg Arg Val 415 420 425
430 ttc ctt gtt ggc cat gat tcg ctt caa caa ctc gga att gaa gat act
1464 Phe Leu Val Gly His Asp Ser Leu Gln Gln Leu Gly Ile Glu Asp
Thr 435 440 445 taa caaagatatg attgcaggag cccgggcaaa atttttgact
tattgggtag 1517 * gatgcatcga gctgacacta aaccatgatt ttaccagtta
catacaacgt tttaatgtta 1577 tacggaggag ctcactgttc tagtgttgaa
gggatatcgg cttcttagta ttggatgaat 1637 catcaacaca acctattatt
ttaagtgttc agaacataaa gaggaaatgt agccctgtaa 1697 agactataca
tgggaccatc ataatcgcgg ccgcgaattc 1737 4 446 PRT Nicotiana tabacum 4
Met Arg Gly Asn Lys Phe Cys Cys Asp Phe Arg Tyr Leu Leu Ile Leu 1 5
10 15 Ala Ala Val Ala Phe Ile Tyr Thr Gln Met Arg Leu Phe Ala Thr
Gln 20 25 30 Ser Glu Tyr Ala Asp Arg Leu Ala Ala Ala Ile Glu Ala
Glu Asn His 35 40 45 Cys Thr Ser Gln Thr Arg Leu Leu Ile Asp Gln
Ile Ser Leu Gln Gln 50 55 60 Gly Arg Ile Val Ala Leu Glu Glu Gln
Met Lys Arg Gln Asp Gln Glu 65 70 75 80 Cys Arg Gln Leu Arg Ala Leu
Val Gln Asp Leu Glu Ser Lys Gly Ile 85 90 95 Lys Lys Leu Ile Gly
Asn Val Gln Met Pro Val Ala Ala Val Val Val 100 105 110 Met Ala Cys
Asn Arg Ala Asp Tyr Leu Glu Lys Thr Ile Lys Ser Ile 115 120 125 Leu
Lys Tyr Gln Ile Ser Val Ala Ser Lys Tyr Pro Leu Phe Ile Ser 130 135
140 Gln Asp Gly Ser His Pro Asp Val Arg Lys Leu Ala Leu Ser Tyr Asp
145 150 155 160 Gln Leu Thr Tyr Met Gln His Leu Asp Phe Glu Pro Val
His Thr Glu 165 170 175 Arg Pro Gly Glu Leu Ile Ala Tyr Tyr Lys Ile
Ala Arg His Tyr Lys 180 185 190 Trp Ala Leu Asp Gln Leu Phe Tyr Lys
His Asn Phe Ser Arg Val Ile 195 200 205 Ile Leu Glu Asp Asp Met Glu
Ile Ala Pro Asp Phe Phe Asp Phe Phe 210 215 220 Glu Ala Gly Ala Thr
Leu Leu Asp Arg Asp Lys Ser Ile Met Ala Ile 225 230 235 240 Ser Ser
Trp Asn Asp Asn Gly Gln Met Gln Phe Val Gln Asp Pro Tyr 245 250 255
Ala Leu Tyr Arg Ser Asp Phe Phe Pro Gly Leu Gly Trp Met Leu Ser 260
265 270 Lys Ser Thr Trp Asp Glu Leu Ser Pro Lys Trp Pro Lys Ala Tyr
Trp 275 280 285 Asp Asp Trp Leu Arg Leu Lys Glu Asn His Arg Gly Arg
Gln Phe Ile 290 295 300 Arg Pro Glu Val Cys Arg Thr Tyr Asn Phe Gly
Glu His Gly Ser Ser 305 310 315 320 Leu Gly Gln Phe Phe Lys Gln Tyr
Leu Glu Pro Ile Lys Leu Asn Asp 325 330 335 Val Gln Val Asp Trp Lys
Ser Met Asp Leu Ser Tyr Leu Leu Glu
Asp 340 345 350 Asn Tyr Val Lys His Phe Gly Asp Leu Val Lys Lys Ala
Lys Pro Ile 355 360 365 His Gly Ala Asp Ala Val Leu Lys Ala Phe Asn
Ile Asp Gly Asp Val 370 375 380 Arg Ile Gln Tyr Arg Asp Gln Leu Asp
Phe Glu Asn Ile Ala Arg Gln 385 390 395 400 Phe Gly Ile Phe Glu Glu
Trp Lys Asp Gly Val Pro Arg Ala Ala Tyr 405 410 415 Lys Gly Ile Val
Val Phe Arg Tyr Gln Thr Ser Arg Arg Val Phe Leu 420 425 430 Val Gly
His Asp Ser Leu Gln Gln Leu Gly Ile Glu Asp Thr 435 440 445 5 1854
DNA Arabidopsis thaliana misc_feature (1185)...(1193) function Asn
Codon is a potential glycosylation site; product Consensus sequence
for N-glycosylation; phenotype N glycans modulate protein
characteristics; standard name N glycosylation site; 5 ctcgaggcca
cgaaggccac cgtttttgtt ataacgaacg acaccgtttc aaacaacttc 60
cttattagct agctccctcc cggcggcaaa caccagaaga tccaccgctt ttgatctggt
120 tgtttgtcgt cgat atg gcg agg atc tcg tgt gac ttg aga ttt ctt ctc
170 Met Ala Arg Ile Ser Cys Asp Leu Arg Phe Leu Leu 1 5 10 atc ccg
gca gct ttc atg ttc atc tac atc cag atg agg ctt ttc cag 218 Ile Pro
Ala Ala Phe Met Phe Ile Tyr Ile Gln Met Arg Leu Phe Gln 15 20 25
acg caa tca cag tat gca gat cgc ctc agt tcc gct atc gaa tct gag 266
Thr Gln Ser Gln Tyr Ala Asp Arg Leu Ser Ser Ala Ile Glu Ser Glu 30
35 40 aac cat tgc act agt caa atg cga ggc ctc ata gat gaa gtt agc
atc 314 Asn His Cys Thr Ser Gln Met Arg Gly Leu Ile Asp Glu Val Ser
Ile 45 50 55 60 aaa cag tcg cgg att gtt gcc ctc gaa gat atg aag aac
cgc cag gac 362 Lys Gln Ser Arg Ile Val Ala Leu Glu Asp Met Lys Asn
Arg Gln Asp 65 70 75 gaa gaa ctt gtg cag ctt aag gat cta atc cag
acg ttt gaa aaa aaa 410 Glu Glu Leu Val Gln Leu Lys Asp Leu Ile Gln
Thr Phe Glu Lys Lys 80 85 90 gga ata gca aaa ctc act caa ggt gga
cag atg cct gtg gct gct gta 458 Gly Ile Ala Lys Leu Thr Gln Gly Gly
Gln Met Pro Val Ala Ala Val 95 100 105 gtg gtt atg gcc tgc agt cgt
gca gac tat ctt gaa agg act gtt aaa 506 Val Val Met Ala Cys Ser Arg
Ala Asp Tyr Leu Glu Arg Thr Val Lys 110 115 120 tca gtt tta aca tat
caa act ccc gtt gct tca aaa tat cct cta ttt 554 Ser Val Leu Thr Tyr
Gln Thr Pro Val Ala Ser Lys Tyr Pro Leu Phe 125 130 135 140 ata tct
cag gat gga tct gat caa gct gtc aag agc aag tca ttg agc 602 Ile Ser
Gln Asp Gly Ser Asp Gln Ala Val Lys Ser Lys Ser Leu Ser 145 150 155
tat aat caa tta aca tat atg cag cac ttg gat ttt gaa cca gtg gtc 650
Tyr Asn Gln Leu Thr Tyr Met Gln His Leu Asp Phe Glu Pro Val Val 160
165 170 act gaa agg cct ggt gaa ctg act gcg tac tac aag att gca cgt
cac 698 Thr Glu Arg Pro Gly Glu Leu Thr Ala Tyr Tyr Lys Ile Ala Arg
His 175 180 185 tac aag tgg gca ctg gac cag ttg ttt tac aaa cac aaa
ttt agt cga 746 Tyr Lys Trp Ala Leu Asp Gln Leu Phe Tyr Lys His Lys
Phe Ser Arg 190 195 200 gtg att ata cta gaa gac gat atg gaa att gct
cca gac ttc ttt gat 794 Val Ile Ile Leu Glu Asp Asp Met Glu Ile Ala
Pro Asp Phe Phe Asp 205 210 215 220 tac ttt gag gct gca gct agt ctc
atg gat agg gat aaa acc att atg 842 Tyr Phe Glu Ala Ala Ala Ser Leu
Met Asp Arg Asp Lys Thr Ile Met 225 230 235 gct gct tca tca tgg aat
gat aat gga cag aag cag ttt gtg cat gat 890 Ala Ala Ser Ser Trp Asn
Asp Asn Gly Gln Lys Gln Phe Val His Asp 240 245 250 ccc tat gcg cta
tac cga tca gat ttt ttt cct ggc ctt ggg tgg atg 938 Pro Tyr Ala Leu
Tyr Arg Ser Asp Phe Phe Pro Gly Leu Gly Trp Met 255 260 265 ctc aag
aga tcg act tgg gat gag tta tca cca aag tgg cca aag gct 986 Leu Lys
Arg Ser Thr Trp Asp Glu Leu Ser Pro Lys Trp Pro Lys Ala 270 275 280
tac tgg gat gat tgg ctg aga cta aag gaa aac cat aaa ggc cgc caa
1034 Tyr Trp Asp Asp Trp Leu Arg Leu Lys Glu Asn His Lys Gly Arg
Gln 285 290 295 300 ttc att gca ccg gaa gtc tgt aga aca tac aat ttt
ggt gaa cat ggg 1082 Phe Ile Ala Pro Glu Val Cys Arg Thr Tyr Asn
Phe Gly Glu His Gly 305 310 315 tct agt ttg gga cag ttt ttc agt cag
tat ctg gaa cct ata aag cta 1130 Ser Ser Leu Gly Gln Phe Phe Ser
Gln Tyr Leu Glu Pro Ile Lys Leu 320 325 330 aac gat gtg acg gtt gac
tgg aaa gca aag gac ctg gga tac ctg aca 1178 Asn Asp Val Thr Val
Asp Trp Lys Ala Lys Asp Leu Gly Tyr Leu Thr 335 340 345 gag gga aac
tat acc aag tac ttt tct ggc tta gtg aga caa gca cga 1226 Glu Gly
Asn Tyr Thr Lys Tyr Phe Ser Gly Leu Val Arg Gln Ala Arg 350 355 360
cca att caa ggt tct gac ctt gtc tta aag gct caa aac ata aag gat
1274 Pro Ile Gln Gly Ser Asp Leu Val Leu Lys Ala Gln Asn Ile Lys
Asp 365 370 375 380 gat gat cgt atc cgg tat aaa gac caa gta gag ttt
gaa cgc att gca 1322 Asp Asp Arg Ile Arg Tyr Lys Asp Gln Val Glu
Phe Glu Arg Ile Ala 385 390 395 ggg gaa ttt ggt ata ttt gaa gaa tgg
aag gat ggt gtg cca cga aca 1370 Gly Glu Phe Gly Ile Phe Glu Glu
Trp Lys Asp Gly Val Pro Arg Thr 400 405 410 gca tat aaa gga gta gtg
gtg ttt cga atc cag aca aca aga cgt gta 1418 Ala Tyr Lys Gly Val
Val Val Phe Arg Ile Gln Thr Thr Arg Arg Val 415 420 425 ttc ctg gtt
ggg cca gat tct gta atg cag ctt gga att cga aat tcc 1466 Phe Leu
Val Gly Pro Asp Ser Val Met Gln Leu Gly Ile Arg Asn Ser 430 435 440
tga tgcaaaacat atgaaaggaa aagaagattt tggaccgcat gcagcctcct 1519 *
tctagcagct gttaggttgt attgttattt atggatgagt ttgtagagcg gtggggttaa
1579 ctttaacagc aaggaagctc tggtgaccag gctgattggc ttagaagtta
tgggaacccc 1639 ttgaaagggt cagggttaaa tatatttcag ttgttttatt
agtgattatc ttgtgggtaa 1699 cttatacgaa tgcaaatcat tctatgcagt
ttttcttcgt cccacttgtt ttggcttctc 1759 tattgctagt gtacatatct
cttcaaacat gtactaaata atgcgtgttg cttcaaagaa 1819 gtaactttta
ttaaaaaaaa aaaaaaaaac tcgag 1854 6 444 PRT Arabidopsis thaliana 6
Met Ala Arg Ile Ser Cys Asp Leu Arg Phe Leu Leu Ile Pro Ala Ala 1 5
10 15 Phe Met Phe Ile Tyr Ile Gln Met Arg Leu Phe Gln Thr Gln Ser
Gln 20 25 30 Tyr Ala Asp Arg Leu Ser Ser Ala Ile Glu Ser Glu Asn
His Cys Thr 35 40 45 Ser Gln Met Arg Gly Leu Ile Asp Glu Val Ser
Ile Lys Gln Ser Arg 50 55 60 Ile Val Ala Leu Glu Asp Met Lys Asn
Arg Gln Asp Glu Glu Leu Val 65 70 75 80 Gln Leu Lys Asp Leu Ile Gln
Thr Phe Glu Lys Lys Gly Ile Ala Lys 85 90 95 Leu Thr Gln Gly Gly
Gln Met Pro Val Ala Ala Val Val Val Met Ala 100 105 110 Cys Ser Arg
Ala Asp Tyr Leu Glu Arg Thr Val Lys Ser Val Leu Thr 115 120 125 Tyr
Gln Thr Pro Val Ala Ser Lys Tyr Pro Leu Phe Ile Ser Gln Asp 130 135
140 Gly Ser Asp Gln Ala Val Lys Ser Lys Ser Leu Ser Tyr Asn Gln Leu
145 150 155 160 Thr Tyr Met Gln His Leu Asp Phe Glu Pro Val Val Thr
Glu Arg Pro 165 170 175 Gly Glu Leu Thr Ala Tyr Tyr Lys Ile Ala Arg
His Tyr Lys Trp Ala 180 185 190 Leu Asp Gln Leu Phe Tyr Lys His Lys
Phe Ser Arg Val Ile Ile Leu 195 200 205 Glu Asp Asp Met Glu Ile Ala
Pro Asp Phe Phe Asp Tyr Phe Glu Ala 210 215 220 Ala Ala Ser Leu Met
Asp Arg Asp Lys Thr Ile Met Ala Ala Ser Ser 225 230 235 240 Trp Asn
Asp Asn Gly Gln Lys Gln Phe Val His Asp Pro Tyr Ala Leu 245 250 255
Tyr Arg Ser Asp Phe Phe Pro Gly Leu Gly Trp Met Leu Lys Arg Ser 260
265 270 Thr Trp Asp Glu Leu Ser Pro Lys Trp Pro Lys Ala Tyr Trp Asp
Asp 275 280 285 Trp Leu Arg Leu Lys Glu Asn His Lys Gly Arg Gln Phe
Ile Ala Pro 290 295 300 Glu Val Cys Arg Thr Tyr Asn Phe Gly Glu His
Gly Ser Ser Leu Gly 305 310 315 320 Gln Phe Phe Ser Gln Tyr Leu Glu
Pro Ile Lys Leu Asn Asp Val Thr 325 330 335 Val Asp Trp Lys Ala Lys
Asp Leu Gly Tyr Leu Thr Glu Gly Asn Tyr 340 345 350 Thr Lys Tyr Phe
Ser Gly Leu Val Arg Gln Ala Arg Pro Ile Gln Gly 355 360 365 Ser Asp
Leu Val Leu Lys Ala Gln Asn Ile Lys Asp Asp Asp Arg Ile 370 375 380
Arg Tyr Lys Asp Gln Val Glu Phe Glu Arg Ile Ala Gly Glu Phe Gly 385
390 395 400 Ile Phe Glu Glu Trp Lys Asp Gly Val Pro Arg Thr Ala Tyr
Lys Gly 405 410 415 Val Val Val Phe Arg Ile Gln Thr Thr Arg Arg Val
Phe Leu Val Gly 420 425 430 Pro Asp Ser Val Met Gln Leu Gly Ile Arg
Asn Ser 435 440 7 24 DNA Artificial Sequence Primer for cloning
plant genes 7 tgygynwsng cntggmayga yaay 24 8 24 DNA Artificial
Sequence Primer for cloning plant genes 8 ccanccntrn ccngsraara
artc 24 9 14 PRT Artificial Sequence Primer for cloning plant genes
9 Gln Xaa Gln Phe Val Gln Asp Pro Xaa Ala Leu Tyr Arg Ser 1 5 10 10
48 DNA Artificial Sequence Primer for cloning plant genes 10
atcggaaagc ttggatcccc agtggcrgct gtagttgtta tggcttgc 48 11 27 DNA
Artificial Sequence Primer for cloning plant genes 11 ggccccccct
cgaggtcgac ggtatcg 27 12 51 DNA Artificial Sequence Primer for
cloning plant genes 12 gggcctctag actccagagc yactactctt ccttgctgct
ggctaatctt g 51 13 40 DNA Artificial Sequence Primer for cloning
plant genes 13 catggatccc tcgagaagcg tcaggaccag gagtgccggc 40 14 37
DNA Artificial Sequence Primer for cloning plant genes 14
atcccgggat ccgctacgta tcttcaactc caagttg 37
* * * * *