U.S. patent application number 10/637763 was filed with the patent office on 2005-05-12 for dna sequences encoding the subunit chld of plant magnesium chelatases, and determining the activity of plant magnesium chelatases.
Invention is credited to Grafe, Susanna, Grimm, Bernhard, Hanel, Frank, Papenbrock, Jutta, Schmidt, Frank, Streber, Wolfgang.
Application Number | 20050102712 10/637763 |
Document ID | / |
Family ID | 7827820 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050102712 |
Kind Code |
A1 |
Grimm, Bernhard ; et
al. |
May 12, 2005 |
DNA sequences encoding the subunit CHLD of plant magnesium
chelatases, and determining the activity of plant magnesium
chelatases
Abstract
The present invention relates to a nucleic acid molecule which
encodes a protein with the function of a plant Mg chelatase subunit
CHLD or an active fragment thereof; a protein which has the
function of a plant Mg chelatase subunit CHLD or an active fragment
thereof, preferably a recombinant protein; a method of determining
the interaction of plant Mg chelatase subunits, in which a host
cell is transformed with a DNA sequence as claimed in one or more
of claims 1 to 3 and at least with one DNA sequence encoding a
further subunit of Mg chelatase in such a manner that the
interaction of the Mg chelatase gene products leads to a directly
or indirectly, qualitatively or quantitatively measurable signal,
preferably by activating a marker gene, and transgenic plants,
transgenic plant cells, transgenic plant organs, transgenic plant
seeds, transgenic propagation material comprising an abovementioned
nucleic acid molecule.
Inventors: |
Grimm, Bernhard;
(Gatersleben, DE) ; Papenbrock, Jutta; (Hannover,
DE) ; Hanel, Frank; (Jena, DE) ; Grafe,
Susanna; (Jena, DE) ; Schmidt, Frank;
(Frankfurt, DE) ; Streber, Wolfgang; (Bad Soden,
DE) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
7827820 |
Appl. No.: |
10/637763 |
Filed: |
August 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10637763 |
Aug 8, 2003 |
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09403463 |
Oct 21, 1999 |
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6831207 |
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09403463 |
Oct 21, 1999 |
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PCT/EP98/02483 |
Apr 27, 1998 |
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Current U.S.
Class: |
800/278 ; 435/15;
435/6.12; 435/6.13 |
Current CPC
Class: |
C12N 15/8274 20130101;
C12N 15/8269 20130101; C12N 9/00 20130101 |
Class at
Publication: |
800/278 ;
435/015; 435/006 |
International
Class: |
C12Q 001/68; C12Q
001/48; A01H 001/00; C12N 015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 25, 1997 |
DE |
197 17 656.9 |
Claims
1-29. (canceled)
30. A method for identifying inhibitors or activators of plant Mg
chelatase comprising: a) combining a recombinant protein having the
function of a plant Mg chelatase subunit CHLD with the gene
products of the DNA sequences encoding the Mg chelatase subunits I
and H to make plant Mg chelatase; b) measuring catalytic activity
of the Mg chelatase in the absence of an additional chemical
compound; c) measuring catalytic activity of the Mg chelatase in
the presence of at least one additional chemical compound; and d)
comparing the results of (b) and (c), whereby an inhibitor of Mg
chelatase is identified by a decrease in the catalytic activity of
(c), as compared with (b), and an activator of Mg chelatase is
identified by an increase in the catalytic activity of (c), as
compared with (b).
31. The method according to claim 30, wherein the recombinant
protein having the function of a plant Mg chelatase subunit CHLD
comprises SEQ ID NO:2.
32. The method according to claim 30, wherein the recombinant
protein having the function of a plant Mg chelatase subunit CHLD is
purified.
33. The method according to claim 30, wherein step (a) is performed
in intact cells.
34. The method according to claim 33, wherein protein-containing
extracts or Mg-chelatase-containing fractions are isolated from the
intact cells after step (a) and before step (b).
Description
[0001] The present invention relates to the subunit CHLD of plant
magnesium chelatase (Mg chelatase), to DNA sequences encoding the
subunit CHLD of plant Mg chelatase, to processes for preparing the
subunit CHLD of plant Mg chelatase, to processes for determining
the activity of plant Mg chelatase, and transgenic plants which
have been transformed with the Mg chelatase DNA according to the
invention.
[0002] Being photosystem cofactors, chlorophylls play a role in the
conversion of light into chemical energy and are thus required for
plant growth and survival.
[0003] Magnesium is incorporated into porphyrin during chlorophyll
biosynthesis with the aid of a membrane-associated enzyme, namely
Mg chelatase, which is composed of several subunits.
[0004] It has already been disclosed that bacterial Mg chelatase is
composed of three subunits (D, H and I), whose corresponding gene
sequences are termed bchD, bchH and bchl (Burke et al. (1993), J.
Bacteriol. 175, 2414-2422; Coomber et al. (1990), Mol. Microbiol.
4, 977-989; Gibson et al. (1995), Proc. Natl. Acad. Sci. USA, 92,
1941-1844; Jensen et al. (1996), J. Biol. Chem. 271,
16662-16667).
1TABLE 1 List of the genes of known Mg chelatase subunits
Rhodobacter Rhodobacter Synechocystis Arabidopsis Antirrhinum
capsulatus sphaeroides PCC 6803 thaliana majus Burke et al. Coomber
Jensen et al. Koncz et al. Hudson et al. et al. bchD bchD chlD bchH
bchH chlH chlH olive bchI bchI chlI ch42 3Dchl
[0005] As regards plant Mg chelatases, two subunits have been
described to date which seem to correspond to the bacterial Mg
chelatase subunits bchH and bchl (Koncz et al., (1990), EMBO J. 9,
1337-1346; Hudson et al., (1993), EMBO J. 12, 3711-3719; Eibson et
al., (1996), Plant Physiol. 121, 61-71). It is not known as yet
which other subunits participate in the structure of plant Mg
chelatase. No enzyme activity was observed with the two known plant
subunits CHLI and CHLH, neither alone nor together with the known
bacterial subunits of type D (CHLD and BCHD).
[0006] Due to their key position in chlorophyll biosynthesis, plant
Mg chelatase is a radically new starting point for developing a
novel generation of herbicidal compounds with highly specific
activity. In addition, the vitality and/or growth of phototrophic
uni- and multicellular organisms, in particular bacteria, algae and
plants, can be controlled to a high degree by influencing gene
expression (suppression, overexpression) of the natural or modified
(for example genetically engineered) expression products of Mg
chelatase or else by specific Mg chelatase inhibitors.
[0007] The enzymatic activity of plant Mg chelatase was originally
measured on intact chloroplasts (Castelfranco et al., (1979) Arch.
Biochem. Biophys. 192, 592-598; Fuesler et al. (1982) Plant
Physiol. 69, 421-423). Since then, the activity was also determined
on disrupted plastids (Walker et al. (1991) Proc. Natl. Acad. Sci.
88, 5789-5793) and subplastid membrane fractions (Lee, et al.
(1992). Plant Physiol. 99, 1134-1140).
[0008] Surprisingly, there has now been found a DNA which encodes a
subunit of the enzyme plant Mg chelatase. The DNA will subsequently
be termed chlD, and the amino acid sequence CHLD.
[0009] Moreover, it has been found that a subunit CHLD of plant Mg
chelatase together with the subunits CHLI and CHLH is,
surprisingly, suitable for reconstituting a funtionally intact,
i.e. enzymatically active, plant Mg chelatase, so that the plant Mg
chelatase subunit CHLD according to the invention provides novel
test methods (in vivo and in vitro) for plant Mg chelatase
activity.
[0010] The present invention therefore relates to a nucleic acid
molecule which encodes a protein with the function of a plant Mg
chelatase subunit CHLD, preferably a nucleic acid molecule as shown
in SEQ ID No. 1, a biologically active fragment thereof, and
complementary or antisense-sequences.
[0011] The invention relates to plant chlD sequences, preferably
from dicotyledonous plants, especially preferably Solanaceae and,
in particular, Nicotiana tabacum.
[0012] The present invention futhermore relates to a nucleic acid
molecule which encodes a protein in accordance with SEQ ID No. 2
with the function of a plant Mg chelatase subunit CHLD or a
biologically active fragment thereof or it forms a complementary or
antisense sequence to such a nucleic acid molecule.
[0013] A further object of the present invention is a nucleic acid
molecule which is an oligonucleotide of at least 10 nucleotides in
length and which hybridizes specifically with a nucleic acid
molecule which encodes a protein with the function of a plant Mg
chelatase subunit CHLD, preferably SEQ ID No. 1, a fragment thereof
or its complementary or antisense sequences.
[0014] The term "to hybridize specifically" is generally understood
to refer to the characteristic of a single-stranded nucleic acid
molecule to form, together with a complementary nucleic acid
molecule, hydrogen bridges, base pairs and, if appropriate, a
double strand.
[0015] Another object of the present invention is a nucleic acid
molecule which encodes a peptide from the group consisting of SEQ
ID No. 3, SEQ ID No. 4 and SEQ ID No. 7, and their complementary or
antisense sequences.
[0016] The term "recombinant organism" is to be understood as
meaning the cell of an organism which has been modified by altering
its DNA in vitro or by integration of DNA, for example recombinant
yeast, bacterial, algal, insect or plant cells.
[0017] Writing "chl" in small letters is conventionally used for
the genes of Mg chelatase subunits D, I and H, while the
capitalization "CHL" refers to the gene products, i.e.
proteins.
[0018] A further object of the present invention is the use of the
nucleic acid molecules according to the invention for amplifying,
isolating or identifying a nucleic acid molecule encoding a protein
with the function of a plant Mg chelatase subunit CHLD or a
biologically active fragment thereof, in particular of chlD
structural genes or chlD mRNA of other organisms, preferably of
plant or microbial origin.
[0019] The invention furthermore also relates to the use of the
nucleic acid molecules according to the invention for raising
antibodies against their expression products.
[0020] Another object of the invention are non-naturally occurring
chimeric genes comprising a promoter which is functionally fused to
a DNA molecule according to the invention.
[0021] Another object of the invention is a vector, preferably a
recombinant vector, comprising a nucleic acid molecule according to
the invention comprising a chimeric gene comprising a promoter,
which is functionally fused to a DNA molecule according to the
invention.
[0022] Another object of the invention is a recombinant host cell
which expresses a DNA molecule according to the invention, this
host cell preferably being stably transformed with a recombinant
vector comprising a chimeric gene comprising a promoter which is
functionally fused to a nucleic acid molecule according to the
invention or which is transformed by other transformation methods
known to those skilled in the art and expresses a DNA molecule
according to the invention.
[0023] The invention furthermore relates to the use of the nucleic
acid molecules according to the invention for generating transgenic
plants.
[0024] Another object of the invention are transgenic plants,
transgenic plant cells, transgenic plant organs, transgenic plant
seeds, transgenic propagation material comprising a nucleic acid
molecule according to the invention.
[0025] Another object of the invention are methods of generating
transgenic plants, transgenic plant cells, transgenic plant organs,
transgenic plant seeds, transgenic propagation material by means of
recombinant host cell, wherein this host cell is transformed with a
DNA molecule according to the invention.
[0026] The invention also relates to propagation material of the
plants according to the invention, for example fruits, seeds,
tubers, root stocks, seedlings and cuttings.
[0027] Suitable excipient plants for a gene according to the
invention are all agriculturally important monocotyledonous and
dicotyledonous crop plants, preferably maize and other cereals such
as, for example, wheat, rye, barley, panic grasses, oats, cassava
and rice, and also cotton, tobacco, sugar beet, sugar cane,
potatoes, oilseed rape, sunflowers, soya, or fruit and vegetable
species.
[0028] To express the nucleic acid molecules according to the
invention in plant cells, they are linked to regulatory DNA
elements which ensure transcription in plant cells. These include,
in particular, promoters. In general, any promoter which is active
in plant cells is suitable for expression.
[0029] The promoter may be chosen in such a way that expression is
constitutive or only takes place in a certain tissue, at a certain
point in time of the plant's development or at a point in time
determined by external factors. As regards the plant, the promoter
can be homologous or heterologous. Examples of suitable promoters
are the cauliflower mosaic virus 35S RNA promoter and the maize
ubiquitin promoter for constitutive expression, the patatin gene
promoter B35 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for
sink-specific expression (for example potato tubers, beet, tomato
fruit) or a promoter which ensures expression only in
photosynthetically active tissues, for example the ST-LS1 promoter
(Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947;
Stockhaus et al., EMBO J. 8 (1989), 2445-2451), all promoters which
are constitutively active in plastids, for example the psbA
cassette expression signal sequences (Staub & Maliga (1993)
EMBO Journal 12: 601-606; Zoubenko et al. (1994) Nucleic Acids
Research 22: 3819-3824) and the Prn promoter (Svab+Maliga (1993)
Proc. Natl. Acad. Sci. USA 90:913-917) or, for endosperm-specific
expression, the wheat HMG promoter, the USP promoter, the phaseolin
promoter, or promoters from maize zein genes. Furthermore, a
termination sequence may be present which serves for correctly
terminating transcription and for adding a poly-A tail to the
transcript, which is assumed to have a function in stabilizing the
transcripts. Such elements are described in the literature (cf.
Gielen et al., EMBO J. 8 (1989), 23-29) and are, in general,
exchangeable as desired.
[0030] A large number of cloning vectors are available for
preparing the introduction of foreign genes into higher plants, and
these cloning vectors contain a replication signal for E. coli and
a marker gene for the selection of transformed bacterial cells.
Examples of such vectors are pBR322, pUC series, M13mp series,
pACYC184. The desired sequence can be introduced into the vector at
a suitable restriction cleavage site. The plasmid obtained is used
for transforming E. coli cells. Transformed E. coli cells are grown
in a suitable medium and then harvested and lysed. The plasmid is
recovered. Analytical methods for characterizing the plasmid DNA
obtained are, in general, restriction analyses, gel electrophoreses
and other methods of biochemistry and molecular biology. After each
manipulation, the plasmid DNA can be cleaved and DNA fragments
obtained can be linked to other DNA sequences. Each plasmid DNA
sequence can be cloned in the same or other plasmids.
[0031] A large number of techniques is available for introducing
DNA into a plant host cell. These techniques encompass the
transformation of plant cells with T-DNA using Agrobacterium
tumefaciens or Agrobacterium rhizogenes as transformation agent,
protoplast fusion, injection, DNA electroporation, the introduction
of DNA by means of the biolistic method and the like.
[0032] When injecting and electroporating DNA into plant cells, the
plasmid used need not meet any particular requirements. Simple
plasmids such as, for example, PUC derivatives, may be used. If,
however, whole plants are to be generated from such transformed
cells, the presence of a selectable marker gene is required.
[0033] Depending on the method by which genes are introduced into
the plant cell, other DNA sequences may be required. If, for
example, the Ti or Ri plasmid is used for transforming the plant
cell, at least the right hand side limit, but frequently the right
and left hand side limit, of the Ti and Ri plasmid T-DNA must be
linked, as a flanking region, with the genes to be introduced.
[0034] If agrobacteria are used for the transformation, the DNA to
be introduced must be cloned into specific plasmids, viz. either
into an intermediary vector or into a binary vector. The
intermediary vectors can be integrated by homologous recombination
into the Ti or Ri plasmid of the agrobacteria due to sequences
which are homologous to sequences in the T-DNA. The Ti or Ri
plasmid also contains the vir region which is required for
transferring the T-DNA. Intermediary vectors cannot replicate in
agrobacteria. The intermediary vector can be transferred to
Agrobacterium tumefaciens by means of a helper plasmid
(conjugation). Binary vectors are capable of replication both in E.
coli and in agrobacteria. They contain a selection marker gene and
a linker or polylinker which is framed by the left and right T-DNA
border region. They can be transformed directly into the
agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978),
181-187). The agrobacterium which acts as the host cell should
contain a plasmid which carries a vir region. The vir region is
required for transferring the T-DNA into the plant cell; additional
T-DNA may be present. This transformed agrobacterium is used for
transforming plant cells.
[0035] The use of T-DNA for transforming plant cells has been
described extensively in EP 120 516; Hoekema, in: The Binary Plant
Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985),
Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 146 and An et
al. EMBO J. 4 (1985), 277-287.
[0036] To transfer the DNA into the plant cell, plant explants may
advantageously be cocultured with Agrobacterium tumefaciens or
Agrobacterium rhizogenes. Whole plants may be regenerated from the
infected plant material (for example leaf sections, stem segments,
roots, but also protoplasts or suspension-cultured plant cells) in
a suitable medium which may contain antibiotics or biocides for
selecting transformed cells. The resulting plants can then be
tested for the presence of the DNA which has been introduced. Other
possibilities of introducing foreign DNA using the biolistic method
or by protoplast transformation are known (cf., for example,
Willmitzer, L., 1993 Transgenic Plants. In: Biotechnology, A
Multi-Volume Comprehensive Treatise (H. J. Rehm, G. Reed, A.
Puihler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim).
[0037] Alternative systems for the transformation of
monocotyledonous plants are the transformation by means of the
biolistic approach, the electrically or chemically induced uptake
of DNA into protoplasts, the electroporation of partially
permeabilized cells, the macroinjection of DNA into inflorescences,
the microinjection of DNA into microspores and proembryos, the DNA
uptake by germinating pollen, and the DNA uptake in embyros by
swelling (review: Potrykus, Physiol. Plant (1990), 269-273).
[0038] While the transformation of dicotyledonous plants via
Ti-plasmid vector systems with the aid of Agrobacterium tumefaciens
is well established, later publications suggest that even
monocotyledonous plants are indeed accessible to transformation by
means of Agrobacterium-based vectors (Chan et al., Plant Mol. Biol.
22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282;
Bytebier et al., Proc. Natl. Acad. Sci. USA 84 (1987), 5345-5349;
Raineri et al., Bio/Technology 8 (1990), 33-38; Gould et al., Plant
Physiol. 95 (1991), 426-434; Mooney et al., Plant Cell Tiss. &
Org. Cult. 25 (1991), 209-218; Li et al., Plant Biol. 20 (1992),
1037-1048).
[0039] Three of the abovementioned transformation systems were
established in the past for a variety of cereals: the
electroporation of tissue, the transformation of protoplasts, and
the DNA transfer by particle bombardment of regenerable tissue and
cells (Jhne et al., Euphytica 85 (1995), 35-44).
[0040] The transformation of wheat is described in various
references (review: Maheshwari et al., Critical Reviews in Plant
Science 14 (2) (1995), 149-178), cf. also Hess et al. (Plant Sci.
72 (1990), 233), Vasil et al. (Bio/Technology 10 (1992), 667-674),
Weeks et al. (Plant Physiol. 102 (1993), 1077-1084), and Becker et
al. (Plant J. 5 (2) (1994), 299-307).
[0041] Once the DNA introduced is integrated in the genome of the
plant cell, it is, as a rule, stable and is retained in the progeny
of the originally transformed cell. It normally contains a
selection marker which mediates resistance to a biocide such as
phosphinothricin or an antibiotic such as kanamycin, G 418,
bleomycin or hygromycin to the transformed plant cells. The marker,
which is chosen individually, should therefore allow the selection
of transformed cells over cells which lack the DNA introduced.
[0042] The transformed cells grow within the plant in the customary
manner (see also McCormick et al., Plant Cell Reports 5 (1986),
81-84). The resulting plants can be grown normally and hybridize
with plants which have the same transformed genetic material, or
other genetic material. The resulting hybrids have the
corresponding phenotypic characteristics. Seeds may be obtained
from the plant cells.
[0043] Two or more generations should be grown in order to ensure
that the phenotypic trait is stably retained and inherited. Also,
seeds should be harvested to ensure that the relevant phenotype, or
other characteristics, have been retained.
[0044] Another object of the invention are, furthermore, methods of
generating a nucleic acid molecule according to the invention,
wherein a nucleic acid molecule according to the invention is
generated by a method known per se for the generation of nucleic
acids. A wide range of different methods of isolating and cloning
gene sequences are available to those skilled in cDNA cloning.
[0045] In addition, the sequence informations according to the
invention can also be utilized for a multiplicity of other methods
known to those skilled in the art, such as, for example, screening
expression libraries for isolating genes using antibodies, or else
for raising antibodies (polyclonal or monoclonal).
[0046] The entire plant chlD cDNA, preferably chlD from Nicotiana
tabacum as shown in SEQ ID No.1, subsequences from SEQ ID No.1 or
oligonucleotides derived from SEQ ID No.1 may be employed as probes
for such methods, if appropriate those which selectively hybridize
with other CHLD-encoding sequences from a library of cloned gene
fragments of a selected organism, for example phototrophic
microorganisms or mono- or dicotyledonous plants.
[0047] Such techniques include, for example, also the screening of
genomic or cDNA libraries, cloned into suitable cells or viruses,
and the amplification by means of polymerase chain reaction (PCR)
using oligonucleotide primers derived from SEQ ID No. 1.
[0048] The isolated chlD (sub)sequences according to the present
invention can furthermore be modified or extended by standard
methods of genetic engineering so as to obtain desired properties.
To obtain selective hybridization under in-vitro or in-vivo
conditions, chlD-specific probes have a length of at least 10
nucleotides, preferably at least 17 nucleotides.
[0049] Hybridization probes according to the present invention can
be used, for example, to amplify or analyze CHLD-encoding sequences
(i.e. CHLD-encoding nucleic acid molecules) from a selected
organism by means of the PCR method, which is known to those
skilled in the art, and can furthermore be used for isolating
CHLD-encoding sequences from other organisms or as a diagnostic for
detecting CHLD-encoding sequences in other organisms, or to
identify and isolate modified CHLD-encoding sequences of a specific
phenotype, such as, for example, resistance to herbicides, altered
chlorophyll content, altered fitness, altered yield and the
like.
[0050] The chlD-specific hybridizaiton probes can also be used to
map the location of a naturally occurring chlD gene in a plant
genome, using standard methods. These methods include, inter alia,
identification of DNA polymorphisms and the use of such
polymorphisms for analyzing the segregation of chlD genes in
relation to other markers of known location. Mapping chlD genes may
be of particular interest in plant breeding.
[0051] chlD-specific hybridization probes or CHLD-encoding
(sub)sequences may also be exploited to inhibit expression of a
chlD gene in planta (antisense technology).
[0052] Using standard methods, it is also possible to modify
CHLD-encoding (sub)sequences or to extend them by novel sequence
elements and to introduce them into the genome of plants. Plant
transformation can be transient or stable, using conventional
methods. The integration of sequences derived from chlD cDNA into
the plant genome can be exploited for altering the properties of
the plant, so that, for example, more or less functionally active
Mg chelatase or a variant of Mg chelatase with altered properties
is formed in the transgenic plant, or else that the expression
level of the chlD gene in the transgenic plant is reduced. For
example, the amount of functional Mg chelatase can be increased by
standard methods if, for example, the chlD gene, alone or together
with genes of other Mg chelatase subunits, is cloned into the
transgenic plant under the transcriptional control of regulatory
elements such as promoters and enhancers. An increased CHLD
activity can be used, for example, for raising the chlorophyll
content, which may be linked to higher yields, or to raise the
tolerance to herbicides which inhibit chlorophyll biosynthesis.
Also, for example cloning altered sequences according to the
invention into transgenic plants, may be linked with a higher
tolerance to herbicides.
[0053] The nucleic acid molecules which encode chlD sequences or
fragments of these sequences (molecules) and which are introduced
into transgenic plants can, however, also be provided with
regulatory elements such as, for example, promoters and enhancers,
in such a way that this results in lowering the original Mg
chelatase activity in the transgenic plant (for example
cosuppression, formation of chlD antisense RNA). A reduced Mg
chelatase activity can be exploited for generating chlorotic plants
or plants with chlorotic tissue. A reduced chlorophyll content may
be desired, for example, when plants or plant organs are subjected
to industrial processing at a later point in time, or when breeding
for a variety of crops or ornamentals, for example vegetables such
as chicory.
[0054] The present invention furthermore relates to a protein which
has the biological function of a plant Mg chelatase subunit CHLD,
preferably according to SEQ ID No. 2, or a biologically active
fragment thereof.
[0055] Another object of the invention is the use of a protein with
the biological function of a plant Mg chelatase subunit CHLD,
preferably according to SEQ ID No. 2 or a biologically active
fragment thereof, for determining the activity of an Mg chelatase
and for raising antibodies.
[0056] Another object of the invention are methods of generating a
protein with the function of a plant Mg chelatase subunit CHLD in a
recombinant host cell, wherein this host cell is transformed with a
nucleic acid molecule according to the invention encoding a plant
Mg chelatase subunit CHLD or an active fragment thereof, preferably
that a DNA sequence encoding a protein with the function of an Mg
chelatase or a biologically active fragment thereof is inserted
into an expression cassette suitable for the host cell, that the
resulting expression cassette is inserted in a suitable manner into
a vector suitable for the host cell, that a suitable host cell is
transformed with the resulting vector and that the thus transformed
host cell is grown in a suitable medium and that the protein which
is produced by said host cell and which has the function of a plant
Mg chelatase subunit CHLD is isolated in a suitable manner from the
culture medium or from the host cell.
[0057] Yet another object of the present invention is a method of
generating a protein with the function of a plant Mg chelatase or a
biologically active fragment thereof in a recombinant host cell,
wherein this host cell is transformed with a nucleic acid molecule
according to the invention encoding a plant Mg chelatase or an
active fragment thereof, preferably that a DNA sequence encoding a
protein with the function of an Mg chelatase is inserted into an
expression cassette which is suitable for the host cell; that the
resulting expression cassette is inserted in a suitable manner into
a vector suitable for the host cell, that a suitable host cell is
transformed with the resulting vector; and that the host cell thus
transformed is grown in a suitable medium and that the protein
which is produced by said host cell and which has the function of a
plant Mg chelatase is isolated in a suitable manner from the
culture medium or the host cell.
[0058] In this sense, the present invention relates to the
generation of plant Mg chelatase subunit CHLD and of plant Mg
chelatase by genetic engineering. For example, to generate the
proteins according to the invention in a host organism, the DNA
sequences according to the invention can be cloned into an
expression cassette which is suitable for heterologous expression
of the structural gene in the selected host organism.
[0059] Examples of CHLD-encoding DNA sequences which are suitable
for this purpose are the following: plant chlD cDNA, plant chlD
cDNA molecules whose sequence has been altered by customary
methods, but also synthetic DNA sequences which have been derived
from plant chlD cDNA or plant CHLD protein or fragments thereof and
which allow expression of a biologically active Mg chelatase CHLD.
Moreover, it may be desired to introduce, into the expression
cassette, specific regulatory sequences, for example promoters,
operator sequences, enhancers, terminators, signal sequences, 5'-
and 3'-untranslated sequences, or sequences encoding suitable
fusion proteins. The use of regulatory sequences is generally
customary technology, which can be varied within wide limits
depending on the expression strategy. The resulting chlD expression
cassette, provided with the necessary regulatory elements within
the correct reading frame of the chlD structural gene, can be
inserted into an expression vector with which the host organism
selected can be transformed. Suitable expression strategies for
generating recombinant proteins, and corresponding expression
vectors, are generally known for host organisms such as, for
example, Escherichia coli, insect cells and plant cells.
[0060] In general, the term "vector" refers to a suitable vehicle
which is known to those skilled in the art and which allows the
targeted transfer of a single- or double-stranded nucleic acid
molecule into a host cell, for example into a DNA or RNA virus, a
virus fragment, a plasmid construct which can be suitable for
transferring nucleic acids into cells in the presence or absence of
regulatory elements, metal particles as they can be employed, for
example, in the particle-gun method, but it may also include a
nucleic acid molecule which can be directly introduced into a cell
by chemical or physical methods.
[0061] The recombinant organism obtained by stable or transient
transformation for example with a chlD expression cassette can be
used for obtaining recombinant Mg chelatase, preferably CHLD
protein, or cell fractions containing CHLD. However, the
recombinant organism may also directly be a component of an
analytic test system.
[0062] It is also possible for the chlD structural gene together
with the structural genes encoding the two other known subunits of
Mg chelatase, namely CHLH and CHLI, to be introduced into a host
organism with the aid of conventional molecular-genetic processes,
resulting in expression of a functional Mg chelatase in this host
organism.
[0063] A preferred expression system is, for example, the use of
bakers yeast (Saccharomyces cerevisiae) as host organism. Vectors
which may be used are all known yeast vectors which have the
suitable expression signals such as promoters, and suitable
selection markers such as resistance genes or genes which
complement for an auxotrophism.
[0064] The use of plant Mg chelatase is essentially based on its
activity as a heterooligomeric enzyme complex, and this activity is
directly linked to the presence of the subunit CHLD. Providing
functionally intact plant Mg chelatase allows biochemical reactions
to be performed not only in vitro (for example cell-free test
system of Mg chelatase), but also in vivo, for example in uni- or
multicellular recombinant organisms or cell cultures, in particular
yeasts, bacteria, algae, insect cells or plants, and is therefore
not limited to phototrophic organisms.
[0065] On the one hand, these reactions can be exploited for
generating Mg-tetrapyrroles, on the other hand, these biochemical
reactions can be used for determining, in a test system, the effect
of chemical compounds, but also of heterogenous substance mixtures,
relative to the function of Mg chelatase.
[0066] The present invention therefore furthermore relates to a
method of determining the interaction between plant Mg chelatase
subunits, which comprises transforming a host cell with a DNA
sequence encoding a protein with the function of a subunit CHLD or
with a biologically active fragment thereof and at least with one
DNA sequence encoding a further subunit of Mg chelatase in such a
manner that the interaction of the Mg chelatase gene products leads
to a signal which can be measured directly or indirectly,
qualitatively or quantitatively, preferably by activating a marker
gene.
[0067] The method is suited for finding specific inhibitors or
activators of plant Mg chelatase, allowing, inter alia, substances
to be identified which have a potential herbicidal,
growth-inhibitory or -enhancing or phytosanitary action.
[0068] The principle of these cellular (in-vivo) test systems is
based on the fact that a recombinant organism which has been
transformed with structure genes of plant Mg chelatase CHLD and
which functionally expresses one or both of the other subunits of
plant Mg chelatase indicates an essential function of one or more
subunits in a manner which allows substances to be found which
influence this function.
[0069] One function of Mg chelatase subunits consists in
interacting with each other. This interaction leads to the
formation of an enzyme complex and is a prerequisite for the enzyme
function of Mg chelatase.
[0070] The present invention therefore relates to a test system
which allows interaction of Mg chelatase subunit CHLD with one or
two other subunits of the enzyme to be detected, and quantitatively
determined, in vivo.
[0071] For example, interaction of subunit CHLD with the subunit
CHLI can be measured in such a way that both subunits are linked
with two other polypeptides or proteins in such a way that
interaction of the subunits directly or indirectly causes a
reaction in the cell which can be detected qualitatively and/or
quantitatively.
[0072] Polypeptides which are suitable for this purpose are, for
example, domains or subunits of regulatory proteins, for example
from cellular signal transduction or transcription processes whose
function is mediated via a protein-protein interaction.
[0073] Suitable are, for example, regulatory proteins of DNA
transcription where the protein-protein interaction of two or more
subunits can be detected with the aid of a reporter gene, so that
the gene product of the reporter gene is formed only when two or
more of the abovementioned subunits interact with each other.
[0074] To generate such a test system, for example one of the two
Mg chelatase subunits to be tested can be fused to the DNA binding
domain, while the other Mg chelatase subunit is fused to the
transcription-activating domain of the regulatory protein. It is
irrelevant here which of the two proteins is fused to which of the
two domains. Thus, CHLD may be linked to the DNA binding domain and
CHLI to the transcription-activating domain. This linkage can be
mediated via the interaction of a further auxiliary factor, for
example a protein-protein interaction or else protein-ligand
interaction such as, for example, streptavidin-biotin. However, a
covalent linkage, for example by fusion of encoding regions of
genes in question, is preferred.
[0075] An example of such a regulatory protein is the gene product
of the Saccharomyces cerevisiae GAL4 gene.
[0076] A host organism, into whose genome an indicator gene with
the relevant promoter had already been integrated, is transformed
with the above-described fusion genes of Mg chelatase subunits.
Such a host organism is distinguished by the fact that it expresses
all functions of the transcription system used, with the exception
of the regulatory protein intended to be expressed as recombinant
protein. If a microorganism is used as the host organism, the
recombinant organism can subsequently be isolated and multiplied as
required using customary methods of microbiology and is therefore
unlimited in its availability for use in the test system. Host
organisms which are preferably used are therefore microorganisms
which can be transformed and grown readily, such as, for example,
yeast species or E. coli.
[0077] Furthermore, a promoter suitable for the transcription
system is linked to a structural gene which encodes an indicator
protein. This recombinant gene is distinguished by the fact that
activation of the promoter directly or indirectly leads to
expression of the indicator protein. Such an indicator protein is
distinguished by the fact that it leads, in the recombinant
organism, to a change which can be detected readily, for example by
catalyzing pigment formation or a chemoluminescence reaction, by
being colored or fluorescent itself, or by contributing to the
growth of the microorganism.
[0078] The use of the described system in which transcription of an
indicator gene is activated has the advantage over the growth test
described further below in which a mutant is complemented with an
Mg chelatase subunit that the former allows a more specific signal
relative to an inhibitor of the enzyme or of an enzyme function,
while the latter test first requires that cell growth inhibitors
with a site of action other than Mg chelatase be excluded by
control experiments. Thus, the present invention prefers the use of
an indicator gene system to the growth test.
[0079] An example to be mentioned of such a system which puts into
reality the abovementioned interactions with the aid of regulatory
proteins of the transcription system is, in particular, the
Matchmaker.TM. two-hybrid system by Clontech, Palo Alto, Calif.,
USA, Catalog No. #K 1605-1, 1995/96, which is hereby incorporated
by reference.
[0080] To this end, plasmids are generated which cause the
expression, in yeast cells, of CHL D, H and I as fusion proteins
with the GAL4 binding domain and the GAL4 activation domain. To
construct the expression plasmids, the DNA sections which encode
the three Mg chelatase subunits are amplified by means of
polymerase chain reaction (PCR).
[0081] The templates used for amplification are the plasmids which
contain the cDNA of the genes in question. The PCR primers used are
synthetic oligonucleotides which are distinguished by the fact that
the sense primer shows homology to the 5' end, while the antisense
primer shows homology to the 3' end of the gene in question. The
primers may also be provided with restriction sites which
facilitate cloning into the vector plasmid.
[0082] The preferred vectors used are the shuttle plasmids of the
MATCHMAKER.TM. two-hybrid system (Clontech): pGBT9 and pAS2 each
contain a gene which encodes the GAL4 activation domain. pGAD424
and pACT2 each contain a gene which encodes the GAL4 DNA binding
domain. pGBT9 and pGAD424, and pAS2 and pACT2, respectively, are in
each case used together in a system. pGBT9 and pGAD424 differ from
pAS2 and pACT2 by the fact that the abovementioned genes are more
weakly expressed in the first two cases than in the last two
cases.
[0083] Each of the subunits encoding Mg chelatase are cloned
individually into the vectors. Recombinant plasmids are introduced
into E. coli by means of transformation, multiplied and isolated.
In this manner, all 12 possible combinations of all three subunits
with all four vectors are performed. The recombinant plasmids are
termed as follows:
[0084] Gene CHL D in vector pAS2 was termed pCBS1148
[0085] Gene CHL D in vector pACT2 was termed pCBS1149
[0086] Gene CHL D in vector pGBT9 was termed pCBS1150
[0087] Gene CHL D in vector pGAD424 was termed pCBS1151
[0088] Gene CHL H in vector pAS2 was termed pCBS1152
[0089] Gene CHL H in vector pACT2 was termed pCBS1153
[0090] Gene CHL H in vector pGBT9 was termed pCBS1154
[0091] Gen CHL H in vector pGAD424 was termed pCBS1155
[0092] Gene CHL I in vector pAS2 was termed pCBS1156
[0093] Gene CHL I in vector pACT2 was termed pCBS1157
[0094] Gene CHL I in vector pGBT9 was termed pCBS1158
[0095] Gene CHL I in vector pGAD424 was termed pCBS1159
[0096] Then, a reporter yeast strain of the two-hybrid system is
transformed with in each case two plasmids. A preferably used
reporter yeast strain is a strain which expresses, as indicator
protein, a GAL4-inducible beta-galactosidase, preferably strain
SFY526 by Clontech. The two plasmids are selected in such a way
that in each case one encodes a subunit in fusion with the DNA
binding domain while the other encodes a subunit in fusion with the
activation domain. Yeast transformation is performed for example by
the method of Klebe et al., 1983, Gene, 25, 333-341. To determine
induction of the indicator gene, a yeast transformed with both
plasmids is grown on solid medium and the activity of
beta-galactosidase is determined after blotting onto filters using
the method of Breedon and Nasmyth (Breedon, L. and Nasmyth, K.
(1985) Regulation of the yeast HO gene. Cold Spring Harbor Symp.
Quant. Biol. 50, 643-650).
[0097] Alternatively, the transformed yeast is grown in liquid
medium and the beta-galactosidase activity is determined in yeast
crude extract after cell disruption, using the method of Munder and
Frurst (Munder, T. and Frurst, P. (1992). Mol. Cell. Biol. 12,
2091-2099).
[0098] To test substances, the recombinant organism is first grown
in a suitable medium. The recombinant cells are then incubated
together with the substance to be tested. The incubation conditions
are selected in such a way that clearly measurable induction of the
indicator gene takes place during the incubation time without the
addition of substances to be tested. This can be effected for
example by the incubation time extending over part of the growth
phase.
[0099] Alternatively, induction may also be effected by expressing
the proteins which play a role in transcription activation, with
the aid of an inducible promoter. Induction of the indicator gene
can be detected using customary detection reactions. If the
indicator gene in the test system is activated, the consequence of
gene activation is that a greater amount of reporter protein is
formed in the host cells, than without activation of the indicator
gene. The test system is preferably selected in such a way that the
reporter protein is produced in at least twice the amount. Increase
in production of the reporter protein by a factor of at least 10 is
especially preferred. If the reporter protein is an enzyme, the
enzyme activity can be determined by customary measuring methods,
for example by colorimetry, fluorimetry or luminometry. For this
purpose, intact cells or extracts therefrom of various degrees of
purification may be incubated with a suitable chromogenic enzyme
substrate. Incubation with the substrate can be affected as early
as during incubation with the substance to be tested, or else
afterwards.
[0100] If a chemical compound interferes with this interaction
between the subunits, for example by binding to an "interface
region" of the subunit, this compound inhibits the oligomerization
process. Thus, this compound can reduce the amount of enzymatically
active Mg chelatase in an organism and thus also have a potential
herbicidal action. Thus, finding herbicidal compounds which inhibit
the formation of the active Mg chelatase enzyme complex is also
possible via the present test system.
[0101] To detect specific interaction of the substance with a
protein of Mg chelatase, the substance must meet the following
conditions:
[0102] 1) In recombinant cells, a significantly lower induction of
the indicator gene must take place after the substance has been
added than in cells without substance added.
[0103] 2) In an analogous transcription system (control system) in
which the interaction of the Mg chelatase subunits was replaced by
a different interaction, inhibition of the indicator reaction must
only be marginally less or not measurable after addition of the
substance. Any type of linkage, for example also covalent bonds of
proteins, may be used as the other interaction.
[0104] If a substance meets the above conditions, it can be
examined for its action in other test systems, for example in
enzymatic tests with Mg chelatase, in plant cell cultures or in
herbological tests on intact plants.
[0105] Moreover, an Mg chelatase gene can be introduced into a
specific mutant of such an organism which is not capable of growth
without function of the Mg chelatase in question, such as, for
example, a photoautotrophic microorganism in which one or more Mg
chelatase subunits have lost their function by mutation. The use of
such a mutant has the particular advantage that growth of the
recombinant organism in a suitable medium can thus be taken as a
measure for the function of Mg chelatase and can thus
quantitatively describe the effect of test substances on Mg
chelatase. The growth test is distinguished by particularly simple
handling and a rapid turnover of substances.
[0106] To this end, the plant chlD gene can be introduced into a
mutant of an organism which is characterized in that it has an
altered phenotype or is not capable of growth under certain culture
conditions without function of the Mg chelatase, preferably without
the function of the CHLD protein. Such a mutant can be generated
for example by first altering, in a microorganism which requires
the activity of an Mg chelatase for phototrophic growth, the
structural genes for its own Mg chelatase in such a way that the
functional enzyme with Mg chelatase activity is no longer formed,
or not in sufficient amounts, so that the resulting organism is no
longer capable of phototrophic growth. Furthermore, the plant
structural genes chll and chlH are expressed in this mutant. By
additionally expressing the plant chlD structural gene, a
functional plant Mg chelatase can be formed in the genetically
engineered organism, and this plant Mg chelatase reimparts to this
organism the capability of phototrophic growth. The growth of such
a recombinant organism is then a direct measure of the plant Mg
chelatase activity. A growth test with this recombinant organism
makes it possible to determine whether the plant Mg chelatase is
inhibited by a chemical compound added to the culture medium. To
this end, the genetically engineered organism is grown in culture
media under phototrophic culture conditions with and without the
compound to be investigated. The chemical compound to be
investigated is preferably employed in concentrations between
10.sup.-9 M and 10.sup.-3 M, especially preferably in
concentrations between 10.sup.-7 M and 10.sup.-4 M.
[0107] To detect specific inhibition of the Mg chelatase by a
chemical compound to be investigated and to exclude other modes of
action as a cause for growth inhibition, the compound must meet the
following criteria:
[0108] 1) Growth of the genetically engineered organisms must be
significantly poorer in the presence of the compound than in
culture medium without the compound. 2) The compound must not
significantly reduce growth of the same organisms under
heterotrophic conditions.
[0109] Like growth, an altered phenotype of a suitably altered
organism may also be used as indicator for catalytic activity of
plant Mg chelatase. Various pigments of phototrophic microorganisms
are made starting from the reaction product of Mg-chelatase,
Mg-protoporphyrin. A recombinant microorganism constructed as
described above which contains, instead of its own Mg chelatase, a
recombinant plant Mg chelatase containing the recombinant CHLD
protein, is only capable of forming its Mg-protoporphyrin-derived
pigments when the plant Mg chelatase has sufficient activity. The
pigmentation of such a recombinant organism is then a direct
measure of plant Mg chelatase activity. Using this recombinant
organism, analysis of the pigment composition makes it possible to
determine whether the plant Mg chelatase is inhibited by a chemical
compound which is added to the culture medium. To this end, the
genetically engineered organism is grown in culture media with and
without the compound to be investigated under culture conditions
under which pigments synthesized from Mg-protoporphyrin are formed.
The chemical compound to be investigated is preferably employed in
concentrations between 10.sup.-9 M and 10.sup.-3 M, especially
preferably in concentrations between 10.sup.-7 M and 10.sup.-4 M.
To detect specific inhibition of the Mg chelatase by a chemical
compound to be investigated and to exclude other modes of action as
a cause for altered pigmentation, the compound must meet the
following criteria:
[0110] 1. The genetically engineered organisms show significantly
lesser amounts of pigments formed starting from Mg-protoporphyrin,
or an altered pigment composition, in the presence of the compound
than in culture medium without the compound.
[0111] 2. The compound must not significantly change pigment
formation of the non-genetically-engineered starting organism under
the same culture conditions.
[0112] If both criteria are met, it can be assumed that the
compound examined is a specific inhibitor of plant Mg chelatase. If
only condition 1, but not condition 2 is met, an enzymatic reaction
of Mg chelatase makes it possible to determine whether the compound
examined is a specific inhibitor of plant Mg chelatase.
[0113] If a substance meets the abovementioned conditions, it can
be examined further for its action in the plant with or without
further biochemical examinations directly either on intact plants
or on suitable plant organs. The customary herbological and
physiological methods may be used for this purpose. An essential
prerequisite for various uses such as, for example, the generation
of a biochemical test system for determining a protein function, is
that the protein to be investigated can be obtained in the
functional state and as pure as possible, i.e. free of interfering
activities. As is the case with all cell proteins, this can be
effected in the case of a plant Mg chelatase by isolating the
individual subunits from plant tissue with the aid of customary
protein purification processes. The present invention states that a
functional plant Mg chelatase contains, besides the subunits CHLH
and CHLI, the subunit CHLD, whose sequence, as an example, is given
for Nicotiana tabacum in SEQ ID No. 2. Isolation of a
heterooligomeric protein whose activity is, moreover, also bound to
a membrane fraction of the plant cell, as is the case with Mg
chelatase, is generally considerably more difficult than isolation
of a soluble or homooligomeric, or monomeric, enzyme since the
protein loses its enzymatic activity in the course of the
purification process, and this can subsequently only be restored by
complicated methods. In addition, the enzymes of chlorophyll
biosynthesis are formed as a function of the type of
differentiation and of the developmental state of the cell.
Moreover, these proteins, as is also the case with Mg chelatase,
only represent a small part of the total cell protein so that
particularly high concentration factors would have to be achieved
when isolating them from plant tissue.
[0114] Due to the difficulties described, it is preferred according
to the invention to use the Mg chelatase-encoding nucleic acid
molecules for generating the recombinant plant Mg chelatase,
especially preferably the recombinant Mg chelatase complex composed
of the proteins CHLD, CHLH and CHLI. The recombinant plant CHLD
protein may be very especially preferably mentioned.
[0115] Plant Mg chelatase generated by means of genetic
engineering, preferably CHLD protein generated by means of genetic
engineering, can be purified using a multiplicity of standard
methods. Whether a method is suitable depends in each case on the
host organism used, the expression strategy and other factors which
are known to those skilled in the art of expression and
purification of the recombinant proteins. In order to purify the
recombinant protein, it may also be fused to other peptide
sequences by altering its gene sequence in the expression cassette
in a suitable manner. Fusion components to be used preferably are
peptides or proteins which, as C- or N-terminal fusions, impart to
the recombinant Mg chelatase subunits an affinity with certain
column materials, or which allow the expression product to be
located at a specific site within or outside of the cell, for
example by means of transit or signal peptides or transit or signal
sequences. Such fusions must not affect the function of Mg
chelatase or must be capable of being cleaved off, for example by
incorporating suitable protease cleavage sites. Examples which may
be mentioned of fusion components are oligohistidine tails, the
Strep-Tag.TM., glutathione-S-transferase (GST) or the
maltose-binding protein (MaIE), without this application being
limited to the fusion components given by way of example or
fragments thereof. To purify an active Mg chelatase complex, it may
also suffice if only one of the subunits existing in the complex is
fused to such a peptide or protein. Moreover, it is also possible
to raise specific antibodies starting from already small amounts of
Mg chelatase generated by a recombinant technology, preferably of
the CHLD protein, and with the aid of these antibodies Mg chelatase
or Mg chelatase subunits can be isolated from recombinant organisms
or else from plants.
[0116] Generation of Mg chelatase, preferably of the CHLD protein,
by means of genetic engineering, and their purification, allow for
the first time a plant enzyme with Mg chelatase activity to be
obtained in various degrees of purity, up to the pure Mg chelatase
complex, composed of the subunits CHLD, CHLH and CHLI, or else the
pure CHLD protein. A possible application for isolated or
concentrated Mg chelatase is, for example, its use in biochemical
test systems for determining the enzyme function.
[0117] The present invention furthermore relates to a method of
determining the activity of plant Mg chelatase, where a protein is
combined with a CHLD function with the gene products of the DNA
sequences encoding for Mg chelatase subunits I and H in such a
manner that the enzymatic activity of the Mg chelatase gene
products leads to a directly or indirectly, qualitatively or
quantitatively measurable signal.
[0118] The inhibitory effect of a chemical compound on Mg chelatase
can be measured via a reduction in the typical catalyst activity of
the enzyme or the complete inactivation of Mg chelatase in the
presence of this compound. To this end, the plant Mg chelatase,
composed of subunits CHLD, CHLH and CHLI, is incubated in a
suitable reaction buffer together with its substrates
protoporphyrin, ATP and Mg.sup.2+.
[0119] By way of preferred reaction conditions, there may be
mentioned a reaction buffer with a pH of between pH 4 and pH 11,
preferably 5-10, in particular 6-9, and reaction temperatures
between 2 and 50.degree. C., preferably 10-40.degree. C. and the
like.
[0120] Quantification of the enzyme inhibition can be effected by a
simple comparison of the catalytic activity of Mg chelatase in the
absence and presence of the chemical compound to be
investigated.
[0121] To determine the Mg chelatase activity, a variety of
biochemical measuring methods may be employed, by means of which
either the formation of the reaction products of the
Mg-chelatase-catalyzed reaction is measured, for example,
Mg-protoporphyrin, ADP or phosphate, or else a decrease in
concentration of the enzyme substrates of Mg chelatase is measured,
for example protoporphyrin, ATP or Mg.sup.2+. A large number of
standard methods for determining Mg chelatase activity are
available to those skilled in the art of performing enzyme tests.
Methods which may preferably be mentioned are those for measuring
the conversion rate of protoporphyrin to Mg-protoporphyrin in the
reaction batch by measuring the different fluorescence properties
of Mg-protoporphyrin and protoporphyrin (for example Walker et al.,
Plant Physiol. Biochem. 30:263-269 (1992)) or by photometric
analysis of the altered absorption resulting from the conversion of
protoporphyrin to Mg-protoporphyrin (for example Gorchein, Biochem.
J. 299:869-874 (1991)) or by quantifying protoporphyrin or
Mg-protoporphyrin by HPLC analysis. Determining the ATPase activity
of Mg chelatase may be mentioned as a further preferred measuring
method, and here it is possible to detect, for example, the
formation of phosphate by a phosphate detection, for example by the
methods based on the system described by Fiske et al., J. Biol.
Chem 66:375-400 (1925). In addition, the inhibitory type of a
chemical compound relative to plant Mg chelatase can be determined
by varying the concentrations of the substrate and the inhibitory
substance, using customary biochemical methods.
[0122] Instead of using purified Mg chelatase, it is also possible
to use intact cells of the recombinant organism which gives
recombinant expression of the three Mg chelatase subunits CHLD,
CHLH and CHLI, or protein-containing extracts from this organism,
or Mg-chelatase-containing fractions from this organism which have
been concentrated to different degrees. Yeast cells may be
mentioned as the preferred recombinant host organism for this
purpose. Alternatively, it is also possible to use a functional Mg
chelatase containing the subunits CHLD, CHLH and CHLI which has
been isolated from plant tissue or plant cell cultures.
[0123] If, with the aid of the biochemical or cell-biological test
systems, it is found that a chemical compound inhibits plant Mg
chelatase in vitro, this compound can be investigated directly for
its action on plants, either on intact plants or on suitable parts
of plants. To this end, a multiplicity of conventional herbological
and physiological methods are available to those skilled in the art
assessing active substances on plants.
[0124] Moreover, the Mg chelatase generated by means of genetic
engineering, preferably an Mg chelatase complex with the subunit
CHLD which has been generated by means of genetical engineering, or
else the free subunit CHLD, can also be used for example for
elucidating the spatial structure of the plant enzyme. Generally
known methods such as, for example, X-ray structural analysis of
protein crystals or NMR spectroscopy may be used for elucidating
the spatial structure. The structure information on Mg chelatase,
preferably on the subunit CHLD, can be used, for example, for
rationally designing novel Mg chelatase inhibitors, and thus
potential herbicides.
[0125] The disclosures in German patent application 197 17 656.9
from which this application claims priority, and in the abstract
accompanying this application are incorporated herein by
reference.
[0126] The examples which follow are intended to illustrate the
invention in greater detail and are in no way to be understood as a
limitation:
[0127] Standard methods such as DNA and RNA isolation, sequence
analysis, restriction, cloning, gel electrophoresis, radio
labeling, Southern and Northern blot were carried out by
conventional methods (Sambrook at al., 1989 Molecular Screening: A
laboratory manual 2.sup.nd ed. Cold Spring Harbour Laboratory Press
N.Y. USA; Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74,
5463-5467).
EXAMPLE 1
Cloning a Tobacco cDNA Which Encodes the Mg Chelatase Subunit
CHLD
[0128] To identify the tobacco chlD cDNA, the peptide sequences
VDASGS (SEQ ID No. 3), TDGRGN (SEQ ID No. 4) and AKGAVM (Seq. ID
No.7) were selected which correspond to the protein sequences
derived from the chlD gene of Synechocystis PCC6803 (Jensen et al.
(1996) J. Biol. Chem. 271, 16662-16667) to prepare the following
mixture of DNA primer sequences:
[0129] 1): GA(CT) GT(ACTG) GA(GA) M(AG) (TA)C(ACGT) GT(ACGT)
[0130] 2): AT (AG)TT (ACGT)CC (ACGT)CG (ACGT)CC (AG)TC (ACGT)G
[0131] 3): GC(ACGT) AA (AG) GG (ACGT) GC (ACGT) GT (ACGT) ATG C
[0132] These primers were used in a polymerase chain reaction (PCR)
to amplify the genomic DNA from the Synechocystis PCC6803: 2 cycles
with in each case 1 min. 940, 2 min. 45.degree. C., 3 min.
72.degree. C., then 28 cycles in each case 30 sec. 94.degree. C.,
90 sec. 60.degree. C., 2 min. 72.degree. C.
[0133] The PCR resulted in a DNA fragment of approx. 270 bp which
was cloned into the cloning vector pCRTMII (Invitrogen, San Diego)
and isolated for further handling by means of a restriction
digest.
[0134] The isolated and .sup.32P[dCTP]-labeled fragment of approx.
270 bp in length was used as a hybridization probe to screen a
lambda ZAP II cDNA library of tobacco (Nicotiana tabacum SR1,
Stratagene) following the instructions.
[0135] The DNA present after a cDNA library screening in the
phagemid p Bluescript SK was isolated and sequenced. The chlD cDNA
sequence identified and shown in SEQ ID No. I contains an open
reading frame 2274 nucleotides in length, and untranslated regions
on the 5' and 3' termini.
[0136] Genomic tobacco DNA was hybridized with radio labeled DNA of
the CHLD gene. To this end, the CHLD gene was excised from the
phagemid p-Bluescript SK using EcoRI and HindIII. The resulting two
fragments of sizes 2069 bp and 426 bp were radio labeled using
.sup.32P(dCTP).
EXAMPLE 2
Generation of chlD Antisense Tobacco Plants
[0137] The tobacco cDNA fragment for CHLD which was obtained
according to Example 1 in pBluescript SK- was excized from the
vector pBluescript SK- using KpnI and XbaI and ligated into the
binary vector BinAR (Hofgen and Willmitzer, Plant Science (1990)
66, 22-230) which had been cleaved with KpnI and XbaI.
[0138] First, E. coli strain DH 5 .alpha. was transformed with the
construct and then Agrobacterium tumefaciens strain GV 2260. Using
the leaf-disk transformation method (Horsch et al., Science 228,
1229-31), young tobacco leaf disks were incubated with the
agrobacteria, and the CHLD antisense gene was stably integrated
into the plant genome.
EXAMPLE 3
Generation of the chlD Sense Tobacco Plants
[0139] To generate the chlD sense tobacco plants, the chlD cDNA was
excized from p Bluescript SK- using SmaI and XbaI and cloned into
the BinAR vector after having been subjected to restriction digest.
To stably integrate it into the tobacco genome, the method
described in Example 2 was chosen.
EXAMPLE 4
Analysis of the Transgenic Tobacco Plants
[0140] 67 sense plants and 56 antisense plants were isolated. The
transgenic plants with antisense or sense genes for CHLD showed
gradually different chlorophyll deficiencies. Chlorotic leaves
showed a variety of variation patterns. There were uniformly
decolorized, yellowish-green leaves, leaves with differently
pigmented spots, and leaves with white areas along the leaf veins
and green intercostal areas. As the leaves aged, they lost
chlorophyll. Plants with reduced chlorophyll content also showed
reduced growth (see FIG. 1a to 1d).
[0141] Moreover, the stable integration of the sense or antisense
chlD genes into the genome of tobacco plants was confirmed by
Southern blot analysis, and the mRNA content of the chlD transcript
was determined by Northern blot analysis. In the antisense plants,
the content is reduced in comparison with the wild type. In
comparison with the wild type, chlD sense plants only have slightly
elevated chlD mRNA contents.
[0142] The Mg chelatase activity was determined for transgenic and
wild-type plants by the method of Lee et al. (1992, Plant Physiol.
99, 1134-1140). The sense and antisense plants always show reduced
enzyme activities.
[0143] The phenomenon of the reduced enzyme activities in the
supposedly CHLD-overexpressed transformants can be explained by
negative dominance. An oversupply of the CHLD subunit causes a
disruption of the finely regulated construction of the Mg chelatase
enzyme complex.
[0144] The protoporphyrin IX and chlorophyll content was determined
in transgenic and wild-type plants (see Table 2). It must be
emphasized that the transformants with sense or antisense CHLD mRNA
show, as a rule, protoporphyrin contents in their young leaves
tested (leaf 3, 5 and 7) which are up to 3-5 times higher than
those of the wild-type plants.
[0145] The determination of the chlorophyll content confirms the
macroscopic phenotype. In individual plants, the content was
reduced by up to 25%.
2TABLE 2 Relative protoprophyrin IX and chlorophyll content in
sense and antisense plants Plant Leaf Protoporphyrin IX [%]
Chlorophyll [%] SNN 3 100 100 5 100 7 100 100 AS7 3 584 25 5 542 7
157 28 AS9 3 135 50 5 120 -- 7 -- 49 AS13 3 160 78 5 102 -- 7 115
78 AS18 3 374 74 5 470 -- 7 295 80 AS21 3 231 95 5 188 -- 7 98 101
S1 3 189 78 5 262 -- 7 297 80 S20 3 423 32 5 293 -- 7 144 25 S22 3
189 117 5 151 -- 7 115 94 S29 3 298 25 5 265 -- 7 137 26 S38 3 257
120 5 127 -- 7 -- 107 SNN = Wild type S = Sense As = Antisense
EXAMPLE 5
Construction of Plasmids for Expressing CHL D, H and I as Fusion
Proteins with the GAL4 Binding Domain and the GAL4 Activation
Domain
[0146] To construct the expression plasmids, the DNA encoding the
three Mg chelatase subunits was amplified by means of polymerase
chain reaction (PCR).
[0147] To amplify the chlD gene, 100 ng of plasmid pNTCHLD were
used as template. The following two oligonucleotides were used as
PCR primers: 5'-TGA CCC GGG GGT AGT GGA ACC TGA AAA ACA ACC-3' as
sense primer with SmaI restriction site, and either 5'-GGC GAA TTC
TCA AGA TTC CTT TAA TGC AGA TAA-3' with EcoRI restriction site or
5'-GCG GTC GAC TCA AGA TTC CTT TAA TGC AGA-3' with SaII restriction
site as antisense primer.
[0148] To amplify the CHLH gene, 100 ng of plasmid pNTCHLH were
used as template. The following two oligonucleotides were used as
PCR primers: 5'-GCT GAT ATC GGC TAT TGG CAA TGG TTT ATT CAC-3' with
EcoRV restriction site as sense primer and 5'-GCG TCG ACA TTT ATC
GAT CGA TTC CCT CAA-3' with SaII restriction site as antisense
primer. To amplify the chll gene, 100 ng of plasmid pNTCHLI were
used as template. The following two oligonucleotides were employed
as PCR primers: 5'-CAG CCC GGG GGG TCC ACT ACT AGG-3' with SmaI
restriction site as sense primer and 5'-CAG GTC GAG GCA CAG TAC AAA
GCC-3' with SaII restriction site as antisense primer.
[0149] The shuttle plasmids of MATCHMAKER.TM. two-hybrid system
(Clontech) were used as vectors: pGBT9 and pAS2 each contain a gene
which encodes the GAL4 activation domain. pGAD424 and pACT2 each
contain a gene which encodes the GAL4 DNA binding domain. pGBT9 and
pGAD424, and pAS2 and pACT2, respectively, are in each case used
together in a system. pGBT9 and pGAD424 differ from pAS2 and pACT2
by the fact that the abovementioned genes are more weakly expressed
in the first two cases than in the last two cases.
[0150] To clone chlD into vector pACT2, the gene was amplified by
means of the sense primer with SmaI cleavage site and the antisense
primer with EcoRI cleavage site and subsequently ligated with the
SmaI- and EcoRI-cut vector.
[0151] To clone chlD into in each case one of vectors pAS2, pGBT9
and pGAD424 the gene was amplified by means of the sense primer
with SmaI cleavage site and the antisense primer with SaII cleavage
site and subsequently ligated with the SmaI- and SaII-cut
vector.
[0152] To clone in each case one of genes chlH and I into in each
case one of vectors pAS2, pGBT9 and pGAD424, the genes were
amplified in each case by means of the sense and the antisense
primer and subsequently ligated with the vectors cut in each case
with SmaI and SaII.
[0153] To clone in each case one of genes chlH and I into vector
pACT2, the genes were amplified by means of the sense and the
antisense primer and subsequently ligated with the SmaI- and
XhoI-cut vector. The ligated DNA was introduced into E. coli DH 5a
by means of transformation, and transformed clones were selected on
agar medium with 100 .mu.g ampicillin/ml. Plasmid DNA was isolated
from the recombinant bacteria and tested for identity by means of
restriction analysis.
[0154] All 12 possible combinations of all three subunits with all
four vectors were made in this manner.
EXAMPLE 6
Determining the Interaction Between Mg Chelatase Subunits by Means
of the Two-Hybrid System in Yeast
[0155] .beta.-Galactosidase liquid assay in yeast (Munder and
Frurst, 1992, Mol. Cell. Biol. 12, 2091-2099) using strain
SFY526+pAS2-chlD+pACT2-chll as example.
[0156] Competent yeast cells strain SFY 526 (Harper et al., 1993,
Cell, 75, 805-816) were made by the method of Klebe (Klebe et al.,
1983, Gene, 25, 333-341) and transformed with the plasmids given in
Example 2. SFY 526 was transformed with all plasmids individually
and in all possible combinations and selected on minimal agar (1.5%
agar, 10% YNB glucose) with an addition of amino acids (adenine 20
mg/l; L-histidine HCl 20 mg/l; L-lysine HCl 30 mg/l; L-leucine 30
mg/l; L-tryptophane 20 mg/l).
[0157] The yeast strain SFY526+pAS2-chlD+pACT2-chll to be measured
was grown in 10 ml of minimal medium (10% of YNB glucose) with an
addition of amino acids required (adenine 20 mg/l; L-histidine HCl
20 mg/l and L-lysine HCl 30 mg/l) overnight at 30.degree. C. and
180 rpm in a sterile 50 ml wide-necked flask with metal cap. The
optical density (OD.sub.600) of this overnight culture was recorded
(Beckman DU 640 spectrophotometer), and the values measured were
between 1.0 and 2.0.
[0158] 100 .mu.l of this culture were tested for
.beta.-galactosidase activity. The total sample volume was approx.
1 ml:
[0159] 100 .mu.l culture
[0160] 700 pI Z buffer+mercaptoethanol
[0161] 50 pI trichloromethane
[0162] 50 .mu.l 0.1% SDS
[0163] A zero-value was examined in parallel:
[0164] 700 .mu.l Z buffer
[0165] 50 .mu.l trichloromethane
[0166] 50 .mu.l 0.1% SDS
[0167] Sample and parallel zero value were each vortexed for 30
seconds, and 160 .mu.l of ONPG solution (4 mg
o-nitrophenyl-.beta.-.sub.D-galactop- yranoside on 1 ml of Z
buffer+mercaptoethanol) was added to each of them. The samples were
then shaken carefully and incubated in a water bath at 30.degree.
C. After one hour, the reaction was quenched by adding 400 .mu.l,
of 1M Na.sub.2CO.sub.3, and the samples were spun for 10 minutes at
13,000 rpm (Heraeus Biofuge fresco, fixed-angle rotor 24). The
supernatant was measured at 420 nm against the zero-value (Beckman
DU 640 spectro-photometer), and the .beta.-galactosidase activity
was calculated:
U=1000E.sub.420 (CVt).sup.-1
[0168] E.sub.420 is the extinction at 420 nm, C is the density of
the cell suspension (OD.sub.600), V is the cell suspension volume
employed, and t is the incubation time.
[0169] The measurement was repeated on two further clones, and the
mean was determined.
[0170] Solutions required: Z buffer 1000 ml:
[0171] 16.1 g Na.sub.2HPO.sub.4x7H.sub.2O
[0172] 5.5 g NaH.sub.2PO.sub.4x7H.sub.2O
[0173] 0.75 g KCl
[0174] MgSO.sub.4x7H.sub.2O
[0175] Z buffer+mercaptoethanol (make freshly)
[0176] 100 ml of Z buffer+270 .mu.l mercaptoethanol
EXAMPLE 7
Reconstitution Assay Mg Chelatase Activity in Yeast (Willows et
al., 1996, Eur. J. Biochem. 235,438-443) Strain
SFY526+pAS2-chlD+pACT2-chlH+pSG28
[0177] Competent cells of strain SFY526+pAS2-chlD+pACT2-chlH were
prepared (Klebe et al.; 1983; Gene, 25, 333-341) and transformed
with pSG28 (p423TEF+chll; p423TEF: Mumberg et al.; 1995; Gene, 156;
119-122). Transformants were selected on minimal agar (10% YNB
glucose; 1.5% agar) with an addition of adenine (20 mg/l) and
L-lysine.multidot.HCl (30 mg/l). 15 ml of a preculture (minimal
medium: 10% YNB glucose+20 mg/l adenine+30 mg/l L-lysine HCl) of
this strain were grown for 24 hours at 30.degree. C. and 180 rpm in
a sterile 50 ml wide-necked flask with metal cap. 150 ml of main
culture (minimal medium: 10% YNB glucose+20 mg/l adenine+30 mg/l
L-lysine HCl) were inoculated with 7.5 ml of preculture and shaken
for 20 hours at 30.degree. C. and 180 rpm using a sterile 500 ml
Erlenmeyer flask with cottonwool plug. The cells were sedimented
for 5 minutes at 5000 rpm (Sigma 4K 10) and resuspended in 1.5 ml
of assay buffer (0.1 M tricine pH 7.9, 0.3 M glycerol, 25 mM
MgCl.sub.2, 4 mM DTT). This cell suspension was subsequently
disrupted ultrasonically for 3.times.30 seconds on ice at low
ultrasonic strength.
[0178] The disrupted cells were sedimented for 15 minutes at 5000
rpm (Sigma 4 K 10) and the supernatant was processed further. The
protein content of this yeast extract was determined using the
BioRad protein assay by the method of Bradford.
[0179] The assay volume was 1 ml. 1 ml of assay buffer contained 1
mg of protein extract of the disrupted yeast culture, 4 mM ATP, 1.5
.mu.M protoporphyrin IX, 50 mM phosphocreatine and 10 U
creatinephosphokinase.
[0180] This incubation mix was incubated for one hour in the dark
at 30.degree. C., and a fluorescence emission spectrum between 500
and 650 nm at an excitation wavelength of 420 nm was then recorded
in a Perkin Elmer luminescence spectrometer LS50B and subjected to
HPLC analysis.
[0181] FIG. 1 (a-d) shows transgenic chlD sense and antisense
tobacco plants.
Sequence CWU 1
1
17 1 2495 DNA Nicotiana tabacum CDS (40)..(2313) coding sequence
for subunit chlD of plant magnesium chelatases 1 catctaaaat
cctaaatcaa aaacttcgat gctataaaa atg ggg ttt tgt tca 54 Met Gly Phe
Cys Ser 1 5 act tca acc ctc cca caa aca tca cta tcc aat tct caa tct
tca aca 102 Thr Ser Thr Leu Pro Gln Thr Ser Leu Ser Asn Ser Gln Ser
Ser Thr 10 15 20 ttc ttc aca tac tta aaa cca tgc cca att cta tcc
tcc aca tat tta 150 Phe Phe Thr Tyr Leu Lys Pro Cys Pro Ile Leu Ser
Ser Thr Tyr Leu 25 30 35 agg ccg gaa cgg cta aaa ttt cgc ctc aga
ata agt gcc act gca act 198 Arg Pro Glu Arg Leu Lys Phe Arg Leu Arg
Ile Ser Ala Thr Ala Thr 40 45 50 att gat tca cct aat ggc gct gtt
gca gta gtg gaa cct gaa aaa caa 246 Ile Asp Ser Pro Asn Gly Ala Val
Ala Val Val Glu Pro Glu Lys Gln 55 60 65 cct gag aaa att tcc ttt
ggt aga cag tat ttt cct cta gct gct gtt 294 Pro Glu Lys Ile Ser Phe
Gly Arg Gln Tyr Phe Pro Leu Ala Ala Val 70 75 80 85 att gga cag gat
gct att aaa act gct ctt tta ctt ggg gcc att gac 342 Ile Gly Gln Asp
Ala Ile Lys Thr Ala Leu Leu Leu Gly Ala Ile Asp 90 95 100 cgt gag
ata gga gga att gca ata tgt ggg aag cgt gga aca gcg aaa 390 Arg Glu
Ile Gly Gly Ile Ala Ile Cys Gly Lys Arg Gly Thr Ala Lys 105 110 115
acg tta atg gca cgt gga ttg cat gct att ctg cca cca att gaa gta 438
Thr Leu Met Ala Arg Gly Leu His Ala Ile Leu Pro Pro Ile Glu Val 120
125 130 gtt gtt ggc tca atg gca aat gct gat ccg aac tgt ccc gat gag
tgg 486 Val Val Gly Ser Met Ala Asn Ala Asp Pro Asn Cys Pro Asp Glu
Trp 135 140 145 gaa gac ggg cta gct gac aga gca gaa tat ggg tct gat
ggt aat atc 534 Glu Asp Gly Leu Ala Asp Arg Ala Glu Tyr Gly Ser Asp
Gly Asn Ile 150 155 160 165 aag acc cag ata gtt aaa tcc cca ttt gtt
cag att ccc ctt ggt gtc 582 Lys Thr Gln Ile Val Lys Ser Pro Phe Val
Gln Ile Pro Leu Gly Val 170 175 180 aca gaa gat aga ttg att ggc tct
gtt gat gtc gag gag tcc gtg aaa 630 Thr Glu Asp Arg Leu Ile Gly Ser
Val Asp Val Glu Glu Ser Val Lys 185 190 195 tct gga acc act gtc ttt
caa cca ggc ctc ctc gca gaa gct cat cga 678 Ser Gly Thr Thr Val Phe
Gln Pro Gly Leu Leu Ala Glu Ala His Arg 200 205 210 gga gtt cta tat
gtt gat gag att aat cta tta gat gaa ggt ata agt 726 Gly Val Leu Tyr
Val Asp Glu Ile Asn Leu Leu Asp Glu Gly Ile Ser 215 220 225 aac cta
ctt ctg aat gta ttg act gag gga gtc aat att gta gaa aga 774 Asn Leu
Leu Leu Asn Val Leu Thr Glu Gly Val Asn Ile Val Glu Arg 230 235 240
245 gag gga atc agc ttt cga cat cca tgc aaa cca cta cta att gct acc
822 Glu Gly Ile Ser Phe Arg His Pro Cys Lys Pro Leu Leu Ile Ala Thr
250 255 260 tat aac cct gaa gag ggt gcg gtt cgt gag cat ctg cta gac
cgt att 870 Tyr Asn Pro Glu Glu Gly Ala Val Arg Glu His Leu Leu Asp
Arg Ile 265 270 275 gcg att aat tta agt gca gat ctt cca atg agt ttt
gac gat cgt gtt 918 Ala Ile Asn Leu Ser Ala Asp Leu Pro Met Ser Phe
Asp Asp Arg Val 280 285 290 gca gct gtt gac ata gca aca cgt ttt cag
gag tgt agc aat gag gtt 966 Ala Ala Val Asp Ile Ala Thr Arg Phe Gln
Glu Cys Ser Asn Glu Val 295 300 305 ttt aaa atg gtg gat gaa gaa aca
gac agt gca aaa acc cag ata ata 1014 Phe Lys Met Val Asp Glu Glu
Thr Asp Ser Ala Lys Thr Gln Ile Ile 310 315 320 325 ttg gca agg gag
tat tta aag gat gtc aca atc agt aga gat caa cta 1062 Leu Ala Arg
Glu Tyr Leu Lys Asp Val Thr Ile Ser Arg Asp Gln Leu 330 335 340 aaa
tac ttg gtc atg gaa gca att cgt ggt ggc tgc cag ggg cac cga 1110
Lys Tyr Leu Val Met Glu Ala Ile Arg Gly Gly Cys Gln Gly His Arg 345
350 355 gct gaa ctt tat gct gct cgt gta gcc aaa tgc tta gct gcc atc
gat 1158 Ala Glu Leu Tyr Ala Ala Arg Val Ala Lys Cys Leu Ala Ala
Ile Asp 360 365 370 gga cgt gaa aaa gtt ggt gtt gat gag ctg aaa aaa
gct gta gag ctt 1206 Gly Arg Glu Lys Val Gly Val Asp Glu Leu Lys
Lys Ala Val Glu Leu 375 380 385 gtc atc ctc cca cgt tca act ata gtt
gaa aac cca cca gac cag caa 1254 Val Ile Leu Pro Arg Ser Thr Ile
Val Glu Asn Pro Pro Asp Gln Gln 390 395 400 405 aac cag cag cca cct
cct ccc cct ccc cct ccc caa aat caa gat tct 1302 Asn Gln Gln Pro
Pro Pro Pro Pro Pro Pro Pro Gln Asn Gln Asp Ser 410 415 420 tca gaa
gag cag aat gaa gaa gaa gaa aaa gaa gaa gaa gat caa gag 1350 Ser
Glu Glu Gln Asn Glu Glu Glu Glu Lys Glu Glu Glu Asp Gln Glu 425 430
435 gat gag aaa gat aga gaa aat gaa cag caa cag cca caa gtc cct gat
1398 Asp Glu Lys Asp Arg Glu Asn Glu Gln Gln Gln Pro Gln Val Pro
Asp 440 445 450 gag ttt att ttt gat gcg gaa ggt ggt tta gtg gat gaa
aaa ctt ctc 1446 Glu Phe Ile Phe Asp Ala Glu Gly Gly Leu Val Asp
Glu Lys Leu Leu 455 460 465 ttc ttt gca caa caa gca caa aga cgc aaa
gga aaa gct gga cga gca 1494 Phe Phe Ala Gln Gln Ala Gln Arg Arg
Lys Gly Lys Ala Gly Arg Ala 470 475 480 485 aag aag gtc atc ttt tcc
gaa gat aga ggt cga tat ata aag cca atg 1542 Lys Lys Val Ile Phe
Ser Glu Asp Arg Gly Arg Tyr Ile Lys Pro Met 490 495 500 ctt cca aag
ggt cca gtg aag aga ttg gca gtt gat gca act cta aga 1590 Leu Pro
Lys Gly Pro Val Lys Arg Leu Ala Val Asp Ala Thr Leu Arg 505 510 515
gca gcg gca cca tat cag aag tta cga aga gca aag gac atc caa aaa
1638 Ala Ala Ala Pro Tyr Gln Lys Leu Arg Arg Ala Lys Asp Ile Gln
Lys 520 525 530 act cgc aag gtt tat gta gag aaa act gac atg aga gcc
aaa aga atg 1686 Thr Arg Lys Val Tyr Val Glu Lys Thr Asp Met Arg
Ala Lys Arg Met 535 540 545 gca cgc aaa gcc gga gct ctg gtg ata ttc
gta gtt gac gct agt ggg 1734 Ala Arg Lys Ala Gly Ala Leu Val Ile
Phe Val Val Asp Ala Ser Gly 550 555 560 565 agt atg gca ctg aat aga
atg cag aat gcc aaa gga gca gca ctt aaa 1782 Ser Met Ala Leu Asn
Arg Met Gln Asn Ala Lys Gly Ala Ala Leu Lys 570 575 580 cta ctt gca
gag agt tat aca agc aga gat cag gtc tgt atc att ccc 1830 Leu Leu
Ala Glu Ser Tyr Thr Ser Arg Asp Gln Val Cys Ile Ile Pro 585 590 595
ttc cgc gga gat gct gct gaa gtt ttg ttg cca cct tct agg tca ata
1878 Phe Arg Gly Asp Ala Ala Glu Val Leu Leu Pro Pro Ser Arg Ser
Ile 600 605 610 tcg atg gca aga aat cgt ctt gag aga ctt ccc tgt gga
ggg ggt tct 1926 Ser Met Ala Arg Asn Arg Leu Glu Arg Leu Pro Cys
Gly Gly Gly Ser 615 620 625 ccc ctt gct cat ggg ctt acg acg gca gtt
aga gtt gga atg aat gca 1974 Pro Leu Ala His Gly Leu Thr Thr Ala
Val Arg Val Gly Met Asn Ala 630 635 640 645 gaa aag agt ggt gat gtt
gga cgt atc atg att gtt gca att act gat 2022 Glu Lys Ser Gly Asp
Val Gly Arg Ile Met Ile Val Ala Ile Thr Asp 650 655 660 ggt aga gct
aac atc tct ctt aaa aga tcc aca gac cct gaa gct gaa 2070 Gly Arg
Ala Asn Ile Ser Leu Lys Arg Ser Thr Asp Pro Glu Ala Glu 665 670 675
gct tct gat gca ccc aga cct tct tcc caa gag ctg aag gat gag att
2118 Ala Ser Asp Ala Pro Arg Pro Ser Ser Gln Glu Leu Lys Asp Glu
Ile 680 685 690 ctc gag gtg gct ggt aaa ata tac aaa aca gga atg tct
ctc ctc gtc 2166 Leu Glu Val Ala Gly Lys Ile Tyr Lys Thr Gly Met
Ser Leu Leu Val 695 700 705 ata gat aca gaa aat aag ttt gtt tct act
ggt ttt gcg aaa gaa atc 2214 Ile Asp Thr Glu Asn Lys Phe Val Ser
Thr Gly Phe Ala Lys Glu Ile 710 715 720 725 gcg aga gta gct caa ggg
aag tac tat tat tta cca aat gct tca gat 2262 Ala Arg Val Ala Gln
Gly Lys Tyr Tyr Tyr Leu Pro Asn Ala Ser Asp 730 735 740 gct gtg ata
tct gca gca aca aag gat gca tta tct gca tta aag gaa 2310 Ala Val
Ile Ser Ala Ala Thr Lys Asp Ala Leu Ser Ala Leu Lys Glu 745 750 755
tct tgacctaaac tcgatcgaat taattgtaaa tgttgttttg agtatagatt 2363 Ser
attgggagga tataagagct tgcttgataa ttcttatctt ttgttgtact aattgaactt
2423 atttctcaat tatgcaatca gggtaatgaa gattcttttc atttcaaaaa
aaaaaaaaaa 2483 aaaggaattc ga 2495 2 758 PRT Nicotiana tabacum 2
Met Gly Phe Cys Ser Thr Ser Thr Leu Pro Gln Thr Ser Leu Ser Asn 1 5
10 15 Ser Gln Ser Ser Thr Phe Phe Thr Tyr Leu Lys Pro Cys Pro Ile
Leu 20 25 30 Ser Ser Thr Tyr Leu Arg Pro Glu Arg Leu Lys Phe Arg
Leu Arg Ile 35 40 45 Ser Ala Thr Ala Thr Ile Asp Ser Pro Asn Gly
Ala Val Ala Val Val 50 55 60 Glu Pro Glu Lys Gln Pro Glu Lys Ile
Ser Phe Gly Arg Gln Tyr Phe 65 70 75 80 Pro Leu Ala Ala Val Ile Gly
Gln Asp Ala Ile Lys Thr Ala Leu Leu 85 90 95 Leu Gly Ala Ile Asp
Arg Glu Ile Gly Gly Ile Ala Ile Cys Gly Lys 100 105 110 Arg Gly Thr
Ala Lys Thr Leu Met Ala Arg Gly Leu His Ala Ile Leu 115 120 125 Pro
Pro Ile Glu Val Val Val Gly Ser Met Ala Asn Ala Asp Pro Asn 130 135
140 Cys Pro Asp Glu Trp Glu Asp Gly Leu Ala Asp Arg Ala Glu Tyr Gly
145 150 155 160 Ser Asp Gly Asn Ile Lys Thr Gln Ile Val Lys Ser Pro
Phe Val Gln 165 170 175 Ile Pro Leu Gly Val Thr Glu Asp Arg Leu Ile
Gly Ser Val Asp Val 180 185 190 Glu Glu Ser Val Lys Ser Gly Thr Thr
Val Phe Gln Pro Gly Leu Leu 195 200 205 Ala Glu Ala His Arg Gly Val
Leu Tyr Val Asp Glu Ile Asn Leu Leu 210 215 220 Asp Glu Gly Ile Ser
Asn Leu Leu Leu Asn Val Leu Thr Glu Gly Val 225 230 235 240 Asn Ile
Val Glu Arg Glu Gly Ile Ser Phe Arg His Pro Cys Lys Pro 245 250 255
Leu Leu Ile Ala Thr Tyr Asn Pro Glu Glu Gly Ala Val Arg Glu His 260
265 270 Leu Leu Asp Arg Ile Ala Ile Asn Leu Ser Ala Asp Leu Pro Met
Ser 275 280 285 Phe Asp Asp Arg Val Ala Ala Val Asp Ile Ala Thr Arg
Phe Gln Glu 290 295 300 Cys Ser Asn Glu Val Phe Lys Met Val Asp Glu
Glu Thr Asp Ser Ala 305 310 315 320 Lys Thr Gln Ile Ile Leu Ala Arg
Glu Tyr Leu Lys Asp Val Thr Ile 325 330 335 Ser Arg Asp Gln Leu Lys
Tyr Leu Val Met Glu Ala Ile Arg Gly Gly 340 345 350 Cys Gln Gly His
Arg Ala Glu Leu Tyr Ala Ala Arg Val Ala Lys Cys 355 360 365 Leu Ala
Ala Ile Asp Gly Arg Glu Lys Val Gly Val Asp Glu Leu Lys 370 375 380
Lys Ala Val Glu Leu Val Ile Leu Pro Arg Ser Thr Ile Val Glu Asn 385
390 395 400 Pro Pro Asp Gln Gln Asn Gln Gln Pro Pro Pro Pro Pro Pro
Pro Pro 405 410 415 Gln Asn Gln Asp Ser Ser Glu Glu Gln Asn Glu Glu
Glu Glu Lys Glu 420 425 430 Glu Glu Asp Gln Glu Asp Glu Lys Asp Arg
Glu Asn Glu Gln Gln Gln 435 440 445 Pro Gln Val Pro Asp Glu Phe Ile
Phe Asp Ala Glu Gly Gly Leu Val 450 455 460 Asp Glu Lys Leu Leu Phe
Phe Ala Gln Gln Ala Gln Arg Arg Lys Gly 465 470 475 480 Lys Ala Gly
Arg Ala Lys Lys Val Ile Phe Ser Glu Asp Arg Gly Arg 485 490 495 Tyr
Ile Lys Pro Met Leu Pro Lys Gly Pro Val Lys Arg Leu Ala Val 500 505
510 Asp Ala Thr Leu Arg Ala Ala Ala Pro Tyr Gln Lys Leu Arg Arg Ala
515 520 525 Lys Asp Ile Gln Lys Thr Arg Lys Val Tyr Val Glu Lys Thr
Asp Met 530 535 540 Arg Ala Lys Arg Met Ala Arg Lys Ala Gly Ala Leu
Val Ile Phe Val 545 550 555 560 Val Asp Ala Ser Gly Ser Met Ala Leu
Asn Arg Met Gln Asn Ala Lys 565 570 575 Gly Ala Ala Leu Lys Leu Leu
Ala Glu Ser Tyr Thr Ser Arg Asp Gln 580 585 590 Val Cys Ile Ile Pro
Phe Arg Gly Asp Ala Ala Glu Val Leu Leu Pro 595 600 605 Pro Ser Arg
Ser Ile Ser Met Ala Arg Asn Arg Leu Glu Arg Leu Pro 610 615 620 Cys
Gly Gly Gly Ser Pro Leu Ala His Gly Leu Thr Thr Ala Val Arg 625 630
635 640 Val Gly Met Asn Ala Glu Lys Ser Gly Asp Val Gly Arg Ile Met
Ile 645 650 655 Val Ala Ile Thr Asp Gly Arg Ala Asn Ile Ser Leu Lys
Arg Ser Thr 660 665 670 Asp Pro Glu Ala Glu Ala Ser Asp Ala Pro Arg
Pro Ser Ser Gln Glu 675 680 685 Leu Lys Asp Glu Ile Leu Glu Val Ala
Gly Lys Ile Tyr Lys Thr Gly 690 695 700 Met Ser Leu Leu Val Ile Asp
Thr Glu Asn Lys Phe Val Ser Thr Gly 705 710 715 720 Phe Ala Lys Glu
Ile Ala Arg Val Ala Gln Gly Lys Tyr Tyr Tyr Leu 725 730 735 Pro Asn
Ala Ser Asp Ala Val Ile Ser Ala Ala Thr Lys Asp Ala Leu 740 745 750
Ser Ala Leu Lys Glu Ser 755 3 6 PRT Synechocystis PCC6803 PEPTIDE
(1)..(6) peptide sequence derived from chlD gene of synechocystis
PCC6803 3 Val Asp Ala Ser Gly Ser 1 5 4 6 PRT Synechocystis PCC6803
PEPTIDE (1)..(6) peptide sequence derived from chlD gene of
synechocystis PCC6803 4 Thr Asp Gly Arg Gly Asn 1 5 5 1579 DNA
Nicotiana tabacum misc_feature (1)..(1579) coding sequence for
subunit chlI of plant magnesium chelatases 5 cccaaaattc ttcttcttct
tcttcactga aaaattctaa acaaaatggc ttcactacta 60 ggaacttcct
cttcagcagc agctgcaata ttagcttcta cacccttgtc ttctcgctcc 120
tgtaagcctg ccgttttctc cctcttccct tcttcagggc agagtcaagg gaggaagttt
180 tatggaggga ttagagtccc agttaagaaa gggaggtccc aattccatgt
ggcaatttca 240 aatgttgcga cggaaatcaa cctgctcaag aacagggtca
gaaacttgct ggaggagagc 300 cagagaccgg tgtatccatt tgcagctata
gtgggacaag atgaaatgaa gttatgtctt 360 ttgctgaatg taattgatcc
aaagattgga ggtgtgatga taatgggtga taggggaacc 420 gggaagtcca
ccacggttag atctttggta gatttacttc ctgaaatcaa agttatttct 480
ggtgatccgt tcaattcaga tccagatgac caagaagtaa tgagtgcaga agtccgtgac
540 aaattgagga gcggacagca gcttcctata tctcgtacga aaatcaacat
ggttgattta 600 ccgcttggtg ctactgagga cagggtgtgt ggcacaatcg
acattgagaa agctcttact 660 gagggtgtga aggctttcga gcctggtctt
cttgctaaag ctaacagagg aatactttac 720 gtcgatgagg ttaatctttt
ggacgaccat ttagtagatg ttcttttgga ttctgcagca 780 tcgggatgga
acactgttga aagagagggg atatcaatat cacaccctgc ccggtttatc 840
cttattggtt cgggtaatcc tgaagaagga gaacttaggc cacaacttct tgatcgattt
900 ggaatgcatg cccaagtggg gaccgtgaga gatgcagagc tgagagtgaa
gatagttgag 960 gaaagagctc gttttgataa gaaccccaag gaattcaggg
aatcatacaa ggcagagcaa 1020 gaaaagctcc agaaccaaat cgactcagct
aggaacgctc tttctgctgt tacaatcgat 1080 catgatcttc gagttaaaat
ctctaaggtc tgtgcagaac taaacgtcga tggattgaga 1140 ggtgatatag
tcactaacag ggcagcaaga gcgttggctg cactaaaagg aagagataag 1200
gtaactccgg aagatatcgc cactgtcatt cccaactgct taagacacag gctgaggaag
1260 gatccgttgg aatctattga ctcgggtgta cttgttgttg agaaatttta
tgaggttttc 1320 gcctaagcgt tttatagagt gagatactta tttttggctt
tattttccat tcataaatca 1380 tctaaagatt tgacaattgt aacactagac
ttttgcttaa ttttggcttt gtactgtgct 1440 taagaaatgg gttcagaatt
acctgtagcc agttgtattt ggttatgact gctttatttc 1500 tgaaatgctt
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560
aaaaaaaaaa aaggaattc 1579 6 4578 DNA Nicotiana tabacum misc_feature
(1)..(4578) sequence for subunit chlH of plant magnesium chelatases
6 agccactact ctccacataa aactataaac ttagaacatt tcacttgaaa aaagagagga
60 aaaaagtgaa gcagaaatct tttctcaaaa cacaatctat aggaagttaa
attcaacttc 120 cacacttcca agattcttgt ttcaagtttc gtttagtttt
tttttcttgg ttttttttat 180 agttttctgt acaattttgt gtagaatcaa
gaaacgaaag agttaaagtt tgaaactttt 240 ttacaagttt gaaacaatgg
cttctttggt ttcttcacca tttacattgc caaattcaaa 300 agtagaacac
ttgtcatcca tttctcaaaa gcattacttt
cttcactcat ttcttcccaa 360 gaaaataaac cccacttact caaaatcacc
aaagaaattc caatgtaatg ctattggcaa 420 tggtttattc actcaaacaa
ctcaagaagt taggagaatt gtgcctgaaa atactcaggg 480 acttgctact
gtgaaaatag tctatgttgt attggaagct cagtaccaat catcacttac 540
tgctgctgtt cagacactga acaagaatgg tcagtttgct tcttttgagg ttgtggggta
600 cttggttgag gagcttagag atgagaatac ttataaaatg ttttgtaaag
atcttgagga 660 tgcaaatgtg tttattggtt cattgatttt tgtggaagaa
ttggctttaa aggtaaaatc 720 tgcagtggag aaagaaaggg acagacttga
tgcagttttg gtgtttccat caatgcctga 780 ggtgatgagg ttgaacaagt
tgggatcttt tagtatgtca caattggggc aatcaaagag 840 tccatttttt
gagcttttca agaagaagaa accttcttct gcaggttttt ctgatcagat 900
gttgaagctt gtgagaacat tgcctaaggt tttgaagtat ttaccaagtg ataaagctca
960 agatgctagg ttgtacatac taagtttgca gttttggcta ggaggttcac
ctgataattt 1020 ggtgaatttc ttgaaaatga tttctggttc ttatgttcct
gctcttaaag ggatgaaaat 1080 cgactactcg gatccggttt tgtacttgga
taatggaatt tggcaccctt tggctccttg 1140 tatgtatgat gatgtgaagg
agtatttgaa ttggtatgca acaaggagag atactaatga 1200 gaaactcaag
agttcaaatg ctcctgttgt tgggctggtt ttgcaaagga gtcatattgt 1260
tacttgtgat gagagtcact atgtggctgt gatcatggaa ttggaggcaa agggggctaa
1320 agttatccca atttttgccg gtgggctaga cttttcgagg ccaattgaga
gatatttcat 1380 tgatcctatt acaaagaagc cttttgtgaa ttcagtaata
tcactttctg gttttgcact 1440 tgttggaggg ccagcaagac aagaccatcc
aagggcaata gaggctttga tgaaacttga 1500 tgtgccttat attgtggcat
tgcctttggt tttccaaaca acagaggaat ggttgaacag 1560 tactttgggg
ctgcacccta ttcaggtggc tctacaagtt gctctccctg agctggatgg 1620
aggaatggag cccatcgtat tcgccggtcg cgatccaaga acagggaaat cacatgctct
1680 tcacaaaaga gtggagcagc tttgcaccag ggcaatcaaa tggggagagt
taaagagaaa 1740 aacaaaggct gagaagaggt tggcaatcac tgtcttcagc
tttcctccag acaaaggcaa 1800 tgtcggaact gctgcatact tgaatgtctt
tgcctccata tactctgttc tcaaagatct 1860 caagaaagac ggctacaacg
ttgaggggct gcctgagact tctgcacaac ttattgaaga 1920 agtaattcac
gacaaagaag ctcagttcag cagcccaaat cttaacatag cttacaagat 1980
gaatgttaga gaataccaga agctaacccc ctatgctact gctcttgaag aaaactgggg
2040 gaaagcacct ggtaatttga actctgatgg agaaaacctc ttggtatatg
gtaaacagta 2100 cggcaatgtc tttatcggtg ttcagcccac gtttggatac
gagggtgacc cgatgagact 2160 tctgttctcc aaatcagcta gccctcacca
tggttttgct gcatactatt cctttgtgga 2220 gaaaattttc aaagctgatg
cagttctcca ctttggtact catggttctc ttgagttcat 2280 gccaggtaaa
caggtgggaa tgagcgatgc ttctttccct gatagtctca ttggaaacat 2340
tcccaatgtc tattactatg cagcaaacaa cccatctgaa gcaactattg ccaaacgaag
2400 gagttatgcg aataccatta gctacttgac tcctccggct gagaatgctg
gactctacaa 2460 gggactcaag cagctcagtg agctcatttc ctcataccaa
tctctgaaag actcaggccg 2520 tggccaacag attgtgaact ctatcatcag
tacagctaga cagtgtaatc ttgacaagga 2580 tgttgatctt ccagaagaag
gggaggaaat ctcggccaaa gagcgtgacc ttgtggtagg 2640 aaaagtatac
tctaagatta tggagatcga gtctcgtctt cttccgtgtg gacttcacat 2700
cattggtgaa cctccaaccg cgatggaagc agttgctact cttgtcaata ttgcgacatt
2760 ggaccgtcct gaagagggta tttctgccct tccatctata ttggctgcga
cggttggaag 2820 aagcattgag gagatttaca gaggcaatga ccagggcatc
ttacgagatg tggagctgct 2880 ccgtcaaatt actgaggcat cacgtggagc
aatatcagca tttgttgaac gtacgacaaa 2940 caacaagggt caggttgtga
atgtcaatga caagctaacc tcaatccttg gttttggtat 3000 aaatgaacca
tggatccagt atttgtcaaa cacccaattt tacagagctg atagggacaa 3060
gctcagagtt ctattccagt tcttgggaga gtgtctgaag ctaattgtcg ctaacaacga
3120 ggtgggaagc ttgaaacagg ctctagaagg gaaatatgtt gaaccaggtc
caggagggga 3180 tccgatcaga aacccgaaag ttttgcctac tgggaaaaac
atccatgctt tggacccaca 3240 agctattccc acaatagcag cagtgcagag
tgccaaaatt gttgttgaaa gattgttgga 3300 gaggcaaaag gccgacaacg
ggggcaagta cccggagact gttgctctgg ttctttgggg 3360 aacagacaac
atcaagacct atggagagtc attggcacag gttatgtgga tgattggtgt 3420
taggccagtt acagactcgt taggacgggt taaccgggtg gaacctgtta gccttgaaga
3480 gcttggaagg cctagagttg atgttgttgt caactgctct ggggtgttca
gagatctctt 3540 catcaatcag atgaatctcc ttgaccgagc agtcaagatg
gttgcagagc tcgacgagcc 3600 agaagaccaa aactacgtca ggaaacatgc
actagaacaa gcaaaaacac tcggagttga 3660 tgttcgtgaa gctgctacaa
ggatcttctc aaatgcttca ggatcttact cctccaacat 3720 taaccttgct
gttgagaatt caacatggaa tgatgagaag caacttcaag acatgtactt 3780
gagccgaaag tcatttgcat ttgactgtga tgcccctggt gttggcatga ctgagaagag
3840 gaaagttttt gagatggctc ttagcacggc tgatgccaca ttccagaacc
ttgactcatc 3900 tgaaatttca ttcacagacg tgagtcacta cttcgattca
gacccaacca accttgtgca 3960 aaacctcagg aaagacggga agaagcctag
tgcatacatt gctgacacca ctactgctaa 4020 tgctcaggta cgtacgttgt
ctgagactgt gaggcttgac gcaaggacaa agttgttgaa 4080 ccccaagtgg
tatgaaggca tgctatccac tggctacgag ggtgttcgtg agattgagaa 4140
acgattaact aacacagtgg ggtggagtgc aacttcaggc caagttgata attgggtgga
4200 tgaagaagcc aacacaacct tcattcaaga tcaggagatg ttgaacaggc
tcatgaacac 4260 aaatccaaat tctttcagga agttgcttca gacattcttg
gaagccaacg ggcgtggata 4320 ctgggaaact tctgcagaga acattgagaa
actcaagcaa ttatactcag aagttgaaga 4380 caagattgag ggaatcgatc
gataaatgta tagcaaaaag aatgatctct gattattgcc 4440 tgtttgttcc
taactgtttc tgatgtgaat tcctttgaca gtccccagtg taattttgtt 4500
catttttggg gatgtcctac ttctatgaga aaatactgct tccatatatt caaatttgag
4560 cttgaaaaaa aaaaaaaa 4578 7 6 PRT Synechocystis PCC6803 PEPTIDE
(1)..(6) peptide sequence derived from chlD gene of synechocystis
PCC6803 7 Ala Lys Gly Ala Val Met 1 5 8 18 DNA Artificial PCR
primer 8 gaygtngara arwcngtn 18 9 19 DNA Artificial PCR primer 9
atrttnccnc gnccrtcng 19 10 19 DNA Artificial PCR primer 10
gcnaarggng cngtnatgc 19 11 33 DNA Artificial Primer 11 tgacccgggg
gtagtggaac ctgaaaaaca acc 33 12 33 DNA Artificial Primer 12
ggcgaattct caagattcct ttaatgcaga taa 33 13 30 DNA Artificial Primer
13 gcggtcgact caagattcct ttaatgcaga 30 14 33 DNA Artificial Primer
14 gctgatatcg gctattggca atggtttatt cac 33 15 30 DNA Artificial
Primer 15 gcgtcgacat ttatcgatcg attccctcaa 30 16 21 DNA Artificial
Primer 16 cagcccgggt ccactactag g 21 17 24 DNA Artificial Primer 17
caggtcgagg cacagtacaa agcc 24
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