Note: Descriptions are shown in the official language in which they were submitted.
CA 02257867 2002-05-24
EDITING~BASED SELECTABLE PLAST7:D MARKER GENES
Pursuant to 35 U.S.C. ~202(c), it is hereby
acknowledged that the U.S. Government has certain
rights in the invention described herein, which was
made in part with funds from the National Science
Foundation.
FIELD OF THE INVENTION
This invention relates to the field of plant
molecular biology. Specifically, DNA constructs are
provided that facilitate the selection of stably
transformed plastids in multicellul;ar plants for
which the encoded RNA is modified post-
transcriptionally.
BACKGROUND OF THE INVENTION
Several publications are referenced in this
application by author names and year of publication
in parenthesis in order to more ful:Ly describe the
state of the art to which this invention pertains.
Full citations for these references are found at the
end of the specification.
Genetic engineering of plants involves the
development and application of technology for genetic
transformation through the direct manipulation of the
plant genome and plant gene expression by the
introduction of novel DNA. One metr~ad of
transformation employs a derivative of ~he tumor
inducing (Ti) plasmid from the bacterium,
Agrobacterium tumefaciens. Other methods utilize
direct gene transfer into protoplasts using
biolistics, electroporation, polyethylene glycol
treatment.
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While the incorporation of transforming DNA in
the nucleus of plant cells is well known to those
skilled in the art, transformation protocols that
selectively identify transformed plastid DNA, to the
exclusion of other genetic compartments have not yet
been described. With the above-described methods, if
plastid transformation only is desired, nuclear
transformants may express the gene encoding the
selectable marker and result in the generation of
to false positives.
The need for plastid-specific marker genes is
based on this observation. In earlier work,
selection for kanamycin resistance of
pTNH32-bombarded tobacco leaves yielded a large
number of nuclear transformants (Carrer et al.,
1993). Indeed, recovery of nuclear gene
transformants with other plastid kan genes
(Cornelissen and Vandewiele 1989), and with
promoterless kan constructs (Koncz et al. 1989)
confirms that kanamycin resistant clones may be
readily obtained by transformation with constructs
that were not designed for expression in the
nucleus. Additionally, nuclear gene transformants in
tobacco may also be recovered by selection for
spectinomycin resistance genes designed for
expression in plastids.
Given the large number of plastid genomes in
plant cells, the ability to select for the
transformed genome in culture is a key element in
achieving successful transformation. Selection
markers have been identified by screening cultured
plant cells for mutants resistant to various
substances, such as antibiotics and herbicides. Such
antibiotics and herbicides are listed in Table I,
below. However, to date, a method has not been
developed that will facilitate plastid transformation
with the concomitant exclusion of the selection of
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nuclear transformants. The development of such a
system minimizes the false positives that result when
a nuclear transformation event occurs.
RNA editing is a process that post-
s transcriptionally alters RNA sequences. Until
recently, it was believed that chloroplasts, in
contrast to mitochondria, did not utilize RNA editing
and that the prediction of amino acid sequences from
the corresponding gene sequences was generally
correct. While most chloroplast genes begin with the
canonical ATG start codon, genes have been identified
that encode an ACG at a position that corresponds to
the 5' terminal ATG in homologous genes in other
species. Recently it has been shown that this ACG
codon is not conserved at the mRNA level. It is
converted to a functional AUG codon by C to U editing
(Hoch et al., 1991). Most of the edited codons found
to date, restore amino acids that are conserved in
the corresponding peptides from chloroplasts of other
species. This editing process is plastid specific.
Genes edited in the plastid are not edii.ed in the
nucleus or other organelles of the plant.
The present invention provides DNA constructs
and methods to facilitate the selection of stably
transformed plastids, based upon a requirement for
RNA editing in the transforming constructs which
occurs exclusively in the plastid. Targeted
manipulation of the plastid genome can now be
performed with greater ease. Such manipulations
include gene replacement, gene deletion, insertion of
foreign genes and expression of recombinant proteins
in plastids.
SUMMARY OF THE INVENTION
This invention provides DNA constructs and
methods for the selection of stably transformed
plastids of multicellular plants. The DNA constructs
CA 02257867 2004-05-26
4
of the invention can be used for the exclusive selection of
plastid transformants. Nuclear transformants will not be selected
with the constructs of the instant invention.
According to one aspect of the invention, there is provided a
recombinant chimeric DNA construct useful for selection of plastid
transformants comprising an edited plastid gene segment selected
from the group consisting of ndhD (SEQ ID NO: 62)and rpl2 (SEQ ID
NO: 64), translationally fused to the coding region of a
selectable marker gene, said selectable marker gene being
expressible following RNA editing of said plastid gene segment.
Cells or tissues are maintained on the selection medium until they
have reached a homoplasmic condition, in which substantially all
of the plastids of the cell or tissue have been transformed.
In a preferred embodiment of the invention, the above
described chimeric construct is incorporated into a vector
containing the necessary homologous sequences for targeted
integration into the plastid genome. The targeting segment is of
sufficient size to promote homologous recombination with a
predetermined plastid genome sequence, thereby replacing that
sequence in the genome of the transformed plastid. The vector may
further comprise a foreign gene of interest to beneficially
augment the phenotype of the plant. In yet another embodiment of
the invention, the chimeric DNA constructs may contain sequences
that direct tissue specific regulation of the foreign gene of
interest.
The method of the present invention is generally applicable
to the selection of stably transformed plastids in both
monocotyledonous and dicotyledonous plants. Following selection,
the cells or tissues expressing the selectable phenotype are
regenerated into multicellular plants.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic of the tobacco plastid
psbE operon and the psbF and psbL DNA and amino acid
5 sequences. The edited psbL initiation codon (from
ACG to AUG) is underlined. The OpsbF/~psbL region is
bounded by dashed lines. The positions of
oligonucleotides 01, 06, 07, 015 and 016 are marked.
The DNA sequence is numbered according to Shinozaki
et al. (1986).
Figure 2 is a physical map and partial DNA
sequence showing, the EaadA gene in plasmid pJLM20,
Figure 2A, and the Ekan gene in plasmid pJLMl8 in
Figure 2B. The conserved -10/-35 promoter elements
and ribosome binding site (RBS) are underlined in the
Prrn sequence. DNA sequence derived from the
OpsbF/~lpsbL region is bounded by dashed lines, new
sequence introduced during construction is in the
solid box. The edited psbL initiation codon (from
ACG to AUG) is underlined. Trpsl6 is the
3'-untranslated region of the plastid rpsl6 ribosomal
protein gene. The positions for oligonucleotides O1,
02 and 03 in EaadA, and for O1, 04 and 05 in Ekan are
indicated. Abbreviation of restriction sites: B,
BspHI; H, HindII3; N, NcoI; S, SacI; X, Xbal.
Figure 3 shows a gel and an autoradiogram
illustrating editing of the EaadA mRNA in the
Nt-pHC94-1 plant, and of the Ekan mRNA in the
Nt-pJLM23-2 plant. Figure 3A shows PCR amplification
products from DNA (lanes 1, 5) and cDNA (lanes 2, 6)
with primers 01 and 02 for EaadA, and primers o1 and
04 for Ekan templates. The location of primers is
a
shown in Figures 2 A & B. Controls were
amplification reactions carried out witr DNase
I-treated RNA (lanes 3, 7) and buffer only (lanes 4
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and 8) using the same primers. Figure 3B shows the
DNA and cDNA sequence of EaadA in the Nt-pHC94-1
plant, and Figure 3C shows Ekan in the Nt-pJLM23-2
plant. The amplified products were directly
sequenced with primers 03 (EaadA) and 05 (Ekan). Due
to the polarity of primers, the sequencE shown is
complementary to the mRNA. The editing site in the
sequence is marked by an arrowhead. Note a mixture
of A and G nucleotides at the editing site in the
cDNA samples indicating partial editing.
Figure 4 depicts a gel and autoradiograms
illustrating editing of the psbL mRNA in the
transgenic plants. Figure 4A depicts the PCR
amplification products from DNA (lanes 1, 5, 9) and
cDNA (lanes 2, 6, 10) from wild-type, Nt-pHC94-1
(EaadA) and Nt-pJLM23-2 (Ekan) plants with primers O1
and 06. See Figure 1. Controls were amplification
reactions carried out with DNase I-treated RNA (lanes
3, 7, 11) and buffer only (lanes 4, 8, 12). The DNA
and cDNA sequence of psbL in wild-type is shown in
Figure 4B. Figure 4C shows the sequences in
Nt-pHC94-1 (EaadA). Figure 4D shows the sequences in
Nt-pJLM23-2 (Ekan) plants. The amplified products
were directly sequenced with primer 07. The sequence
shown is complementary to the mRNA sequence due to
the polarity of the O7 primer. The editing site is
indicated by an arrowhead. Note nearly complete
editing in the wild type (G* is very faint) and
partial editing in the transgenic plants.
Figure 5 shows a partial DNA map of the plastid
genome and an autoradiogram depicting the
steady-state levels of psbL and chimeric mRNAs.
Figure 5A depicts a partial map of the plastid genome
with the EaadA and Ekan genes obtained by
transformation with the pHC94 or pJLM23 plasmids.
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The l6SrDNA and trnV genes, and the rpsi2/7 operon
are marked. Horizontal arrows indicate mRNAs
detected by the 014 oligonucleotide probe. The
autoradiogram in the upper panel in Figure 5B shows
that the 014 oligonucleotide detects the similar size
(1.1-kb) psbE, EaadA and Ekan, and the 2.2-kb
Ekan-aadA transcripts. Additional, minor
uncharacterized RNA species are also visible which
were not included in the quantitation. Total
cellular RNA (2 /.~,g per lane) was loaded from a
wild-type plant (Wt), plasmid pHC94-transformed
plants (Nt-pHC94-1, Nt-pHC94-11, Nt-pHC94-21) and
plasmid pJLM23-transformed plants {Nt-pJLM23-2,
Nt-pJLM23-14, Nt-pJLM23-18). The lower panel in
Figure 5B illustrates the accumulation of psbE mRNA
detected by the psbJ probe, and of the 16S rRNA as
the loading control. The filter was stripped of the
labeled 014 oligonucleotide, and probed with a
mixture of the psbJ and l6SrDNA probes. The psbE
probe was obtained by PCR amplification of the psbJ
region with primers o15 and 016 shown in Figure 1.
The l6SrDNA probe was a 2.4--kb EcoRI/EcoRV ptDNA
fragment defined by the restriction sites at
nucleotides 138448/141847 of the plastid genome
(Shinozaki et al., 1986).
Figure 6 is a series of autoradiograms
illustrating editing of the rpoB and. the ndhB
transcripts in the wild-type and transgenic
Nt-pHC94-1 plants. The DNA and cDNA sequences
corresponding to each gene were PCR amplified using
the following primers: 08 and 09 for rpoB; 010 and
011 for ndhB. The sequencing primer for rpoB was 08,
for ndhB was O1. The editing site in the sequence is
marked by an arrowhead. Arrowhead points at C in DNA
which is edited to T at sites I and II of the rpoB
and ndhB transcripts.
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Figure 7 is a partial map and a series of
autoradiograms illustrating the approach by which the
region required for psbL editing was defined. Figure
7A shows the map of the chimeric OpsbL/kan gene, with
the 98 nt OpsbF/~psb fragment enlarged at the top.
The positions of primers 04, 05 and 017 are
indicated. 66,780 and 66,683 are the nucleotides at
the ends of the 98 nt OpsbF/OpsbL fragm.nt in the
tobacco plastid genome (Shinozaki et al., 1986).
The lower portion of Figure 7A is a listing of
pPRV111A plasmid derivatives which carry chimeric
OpsbL/ka,n genes. The nucleotide position at the end
of the OpsbF/~lpsbL deletion derivatives is given
relative to the edited C (position O; arrow). The
efficiency of editing of the chimeric ~psbL/kan mRNA
(%), and the kanamycin resistance phenotype of the
transgenic plants is listed. Figure 7B is an
autoradiogram demonstrating editing of the psbL site
in the chimeric mRNAs. The cDNAs were PCR amplified
with primers 017 and 04 and directly sequenced with
primer 05. Due to the polarity of 05, :.he sequence
shown is complementary to the mRNA. Accordingly, A at
the edited position indicates a C to U conversion
event and a G an unedited C nucleotide.
Figure 8 is a partial DNA map and a pair of
autoradiograms illustrating psbL editing in
transgenic plants with mutations adjacent to the
editing site. The position of the edited C (arrow)
and the flanking nucleotides within the 98 nt
OpsbF/OpsbL fragment are shown in the upper portion
of Figure 8. Mutations in plasmids pSCl4 and pSCl5
are in lower case. Editing was tested ~y sequencing
the chimeric cDNAs (bottom). Calculated editing
efficiencies of the chimeric mRNA (%) are listed.
For experimental details see legend to Figure 7.
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Figure 9 shows a partial DNA map and a series of
autoradiograms illustrating the existence of
competition for the psbL-specific transfactor
(psbL-SEF) in the transgenic plants. Figure 9A shows
the map of the psbE operon containing the psbL gene,
with the position of oligonucleotides 01, 06 and 07
used for PCR amplification arid sequencing indicated.
The 22 nt (-16/+5) sequence required for editing is
shown. The edited C is marked by an arrow. The 16
nt segment competing for psbL-SEF is boxed. The
plasmids used to obtain the transgenic plants are
listed, as identified in Figures 7 and 8.
Competition (+} was indicated by reduced editing
efficiency of the psbL mRNA, as compared to
nontransformed, wild-type plants. Figure 9B is an
autoradiogram showing editing of psbL mRNAs. The
cDNAs were PCR amplified with primers O1 and 06 arid
directly sequenced with primer 07. Due to the
polarity of 07, the sequence shown is complementary
to the mRNA. Accordingly, A at the edited position
indicates a C to U conversion event and a G at the
edited position, an unedited C nucleotide. A+G*
denotes nearly complete editing (>99%) as in the
wild-type plants. A+G denotes partial editing with
~10% unedited psbL transcripts.
Figure l0 is a partial DNA map and a series of
autoradiograms illustrating that chimeric mRNAs
containing the ndhD editing site do not compete for
psbL-SEF. Figure 10A shows a partial map of the
tobacco plastid genome containing the ndhD, psaC and
ndhE genes, and the DNA sequence with the edited ndhD
translation initiation codon (underlined). The genes
are marked and the DNA sequence is numbered according
to Shinozaki et al., 1986. The llndhD segment i.n a
dashed box was translationally fused with the lean
gene, as shown in Figure 10B. The position of
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primers 018, 029 and 020 are indicated. The A
nucleotide 26 by upstream of the editing site
(underlined) was changed to a C during construction
of the chimeric gene. Figure 10B shows the OndhD/kan
5 chimeric gene in plasmid pSC23 expressed in the
Prrn/Trpsl& cassette. The positions of primers 04,
05 and 017 are indicated. Figure lOC depicts
autoradiograms demonstrating editing of ndhD and psbL
sites in wildtype (Nt-wt), Nt-pSC23 and Nt-pSC2
10 plants. Editing of the ndhD site was studied in the
endogenous ndhD, and the chimeric OndhD/kan mRNAs.
Editing of the psbL site was studied in the
endogenous psbL, and the chimeric OpsbL/kan mRNAs.
The ndhD cDNA was amplified with primers 018 and 019
and sequenced with primer 020. The psbL cDNA was PCR
amplified with primers 01 and 06 and directly
sequenced with primer 07. The ~ndhD/kan and OpsbL/kan
cDNAs were amplified with primers 017 and 04, and
sequenced with 05. Due to the polarity of the
sequencing primers, the sequence shown i.s
complementary to the mRNA. Accordingly, A at the
edited position indicates a C to U conversion event
and a G, an unedited C nucleotide.
Figure 11 is a schematic illustration of the
Glrpl2/kan gene. A partial map of the maize plastid
genome containing the trnI, rp123, rpl2 and trnH
genes, and the DNA sequence with the. edited rpl2
translation initiation codon (underlined) is shown in
Figure 11A. The genes are marked and the DNA
sequence is numbered according to Maier et al., 1995.
The ~rpl2 segment in a dashed box was translationally
fused with the kan gene, as shown in Figure 11B.
Figure 11B demonstrates that the Or-pl2D/kan chimeric
gene in plasmid pSC22 is expressed in the Prrn/Trpsl6
cassette. The position of primers 04, 05 and 017 is
indicated.
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Figure 12 is a schematic drawing of minigenes
used to test editing in segments of ndh~ and rpoB
RNAs. The nhdB plastid gene and its minigene are
shown in the upper portion of Figure 12 and the rpoB
plastid gene and its corresponding minigene (lower
portion of Figure 12) are shown. Editing sites are
indicated in the Figure. Minigene RNAs are expressed
in a cassette which contains the rRNA operon promoter
(Prrn} and the 3' untranslated region of the rpsl6
ribosomal protein gene (Trpsl6) required for mRNA
stability (Zoubenko et al., 1994). The editing sites
and the references are listed in Table IV.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention,
methods and DNA constructs are provided to facilitate
the selection of transformed plastids following
delivery of transforming DNA. The constructs of the
invention will be expressed only if they are
appropriately edited at the RNA level within the
plastid. In so far as it is known, the methods and
DNA constructs described herein have heretofore been
unavailable for multicellular plants.
The following definitions will facilitate the
understanding of the subject matter of the present
invention:
Hetero~lasmia: refers to the presFnce of a
mixed population of different plastid genomes within
a single plastid or in a population of plastids
contained in plant cells or tissues.
Homoplasmia: refers to a pure population of
plastid genomes, either within a plastid or within a
population contained in plant cells and tissues.
Homoplasmic plastids, cells or tissues are
genetically stable because they contain only one type
of plastid genome. Hence, they remain homoplasmic
even after the selection pressure has been removed,
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and selfed progeny are also homoplasmic. For
purposes of the present invention, heteroplasmic
populations of genomes that are functionally
homoplasmic (i.e., contain only minor populations of
wild-type DNA or transformed genomes with sequence
variations) may be referred to herein as
"functionally homoplasmic" or "substantially
homoplasmic.°' These types of cells or tissues can be
readily purified to homoplasmy by continued selection
on the non-lethal selection medium. Most seed
progeny of such plants are homoplasmic in the absence
of selection pressure, due to random sorting of
plastid genomes.
Plastome: the genome of a plastid.
Transplastome: a transformed plastid genome.
Transformation of plastids: stable integration
of transforming DNA into the plastid genome that is
transmitted to the seed progeny of plants containing
the transformed plastids.
selectable marker: the term "selectable marker"
refers to a phenotype that identifies a successfully
transformed organelle, cell or tissue, when a gene or
allele encoding the selectable marker is included in
the foreign DNA used for transformation.
Transforming DNA: refers to homologous DNA, or
heterologous DNA flanked by homologous DNA , which
when introduced into plastids becomes part of the
plastid genome by homologous recombination.
Edited gene segment: refers to a region of DNA
which encodes an RNA which is post-transcriptionally
altered.
Transiationally fused: refers to two coding
regions of two separate genes spliced together in a
construct such that both regions will be expressed at
the protein level. In accordance with the present
invention translation of the chimeric protein is
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dependent on appropriate editing of the upstream
coding region at the mRNA level.
The detailed description as followe provides
examples of preferred methods for making and using
the DNA constructs of the present invention and for
' practicing the methods of the invention. Any
molecular cloning and recombinant DNA techniques not
specifically described are carried out by standard
methods, as generally set forth, for example in
Sambrook et al., "DNA Cloning, A Laboratory Manual,"
Cold Spring Harbor Laboratory, 1989.
In the detailed description and examples set
forth hereinbelow, a preferred embodiment comprises a
DNA segment that encodes an edited RNA segment
operably linked to a second DNA segment which encodes
a selectable marker. In the following examples in
tobacco chloroplasts are exemplified. Transformation
vectors containing such combinations will be useful
in enabling plastid-specific transformation.
References made to positions and sequences on the
tobacco chloroplast genome are taken from Shinozaki
et al., EMBO J., 5: 2043-49 (1986), which discloses
the complete nucleotide sequence of the Nicotiana
tabacum chloroplast genome. Although tobacco is
exemplified, it will be appreciated by those skilled
in the art that the DNA constructs and methods of the
present invention can be adapted to plastids of other
plant species.
Plastid transformation requires: (1) a method
for delivering DNA through the double membrane of the
plastid; (2) integration of the heterologous DNA
without interfering with the normal function of the
plastid genome; and (3) efficient selection for the
transplastome. Methodology for performing efficient
transformation of plastids of multicellular plants is
sat forth in U.S. Patent No. 5,451,513 issued
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September 19, 1995, the entire disclosure of which is
incorporated by reference herein.
In accordance with the present invention, it
has been discovered that the selection criterion for
identifying transplastomes is critical to the success
of stable plastid transformation in higher plants.
Accordingly, the selection technique of the present
invention employs DNA encoding a selectable phenotype
("selectable marker") in the transforming DNA.
Selection greatly facilitates obtaining
transplastomic lines, due in part to the large number
of identical plastid genome copies present in each
plant cell (3,000-12,000 copies localized in up to
100 plastids in tobacco, as compared with 80 copies
carried by a single plastid in Chlamydomonas).
Selectable phenotypes can include antibiotic
resistance, herbicide resistance, drug resistance or
resistance to toxic analogs of metabolites.
The present invention provides selectable marker
genes that require RNA-editing processes which occur
in plastids only, not in the nucleus or in
mitochondria. Such plastid specific marker genes
will greatly enhance the ability to obtain stably
transformed plastids in multicellular plants. The
novel combination of plastid specific editing site
controlling expression of a selectable marker is
hereinafter described.
Certain mRNA sequences can be altered
post-transcriptionally by a process known as RNA
editing, so that their final nucleotide sequence
differs from that encoded by the DNA sequence. The
process has been detected in divergent organisms
including trypanosomes, Physa~um polycephalum,
mammals, viruses and higher plants involving widely
different molecular mechanisms (reviewed in Benne,
1994; Chan, 1993; Gray and Covello, 1993; Innerarity
et al., 1996; Simpson and Thiemann, 1995).
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In higher plants, editing of plastid and
mitochondrial RNAs involves C to U conversions and
rare cases of U to C changes in mitochondria. The
' number of editing sites in plastids is estimated to
5 be about 25 (Maier et al., 1995) while in plant
' mitochondria it is 1000 or more (Schuster and
Brennicke, 1994). Comparison of sequences
surrounding editing sites have failed to identify any
conserved primary sequence and/or structural motifs
10 that could direct the site-selection process. The
recent development of an in vitro editing system
should lead to accelerated progress in the analysis
of RNA editing in plant mitochondria (Araya et al.,
1992; Yu and Schuster, 1995). Although an in vitro
15 system for editing in plastids is still lacking, the
availability of plastid transformation allows an in
vivo approach to study plastid editing (Bock et al.,
1994; Bock and Maliga, 1995; Sutton et al., 1995,.
While RNA editing has been reported to occur in
plastids and mitochondria, it has not been observed
in the nucleus (Kossel et al., 1993; Hanson et al.,
1995). Furthermore, mitochondrial transcripts are
not edited in plastids (Sutton et al., 1995). The
discovery of RNA editing and the problem of
recovering large numbers of nuclear transformants
after bombardment of plant cells with plastid
directed constructs, led to the design of the plastid
transgenes of the invention which are expressed in
plastids but not in other genetic compartments of the
cell. The following examples describe the
transgenes of the invention.
Briefly, one example of an editing based
selectable marker gene utilizes the N-terminal
segment of an edited plastid gene translationally
y
fused to the coding region of an antibiotic
resistance gene. The expression of the antibiotic
resistance gene is dependent upon RNA editing of the
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construct. In a specific example, the ie-terminal
segment of psbL is fused to the coding region of aadA
gene. Translation of the aadA gene is dependent on
editing, and is used to recover plastid transformants
by direct selection (Chaudhuri et al., 1995).
Additionally, a psbL based editing selectable marker
can comprise the similar OpsbL/kan chimeric gene
(Chaudhuri and Maliga, 1996}, in which kanamycin
resistance is a reliable measure of the editing of
the translation initiation codon (Chaudhuri et al.,
1995; Chaudhuri and Maliga, 1996). Although these
genes confer kanamycin resistance to the plant cell
when present in each of the plastid gen~me copies in
a cell, they could not be used for direct selection.
Presumably this is because plastid transformants can
only be directly selected by the kanamycin resistance
marker if the chimeric genes are expressed at high
levels. Direct selection for Ekan genes should be
feasible by improving their expression level through
appropriate engineering.
A second, novel example of an editing based
selectable marker gene utilizes the ndhD edited
segment. This construct, OndhD/kan was obtained by
fusing the N-terminal segment of ndhD and the coding
region of lean (Chaudhuri and Maliga, 2996). The
above described selectable marker genes will
facilitate the selection of transformants in certain
dicot species.
A third type of editing based marker gene,
Oz-p12/kan, is created by fusing the rpl2 edited
segment to the kanamycin coding region. Such
chimeric selectable marker genes will be used to
advantage in selecting plastid transformants in
monocots.
In tobacco plastids, functional psbL and ndhD
mRNA is created by editing an ACG codon to an AUG
translation initiation codon. To determine if
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editing may occur in a chimeric mRNA, the N-terminal
part of psbL containing the editing site was
translationally fused with the aadA and kan bacterial
' genes as described in the following examples. The
chimeric constructs were introduced into the tobacco
' plastid genome by targeted gene insertion. Deletion
derivatives of a 98 pt fragment were expressed as
parts of chimeric transcripts to define the cis
sequences required for psbL editing. In accordance
with the instant invention, it has been found that a
22 pt fragment is sufficient to direct psbL editing.
Although the 22 nucleotides were required for
editing, only 26 nucleotides competed for the
psbL-specific editing factor.
Expression of the chimeric gene transcripts led
to a significant decrease in the editing efficiency
of the endogenous psbL mRNA. However, the efficiency
of editing in the transpiastomic lines was unchanged
for four sites in the rpoB and ndhB mRNAs. Reduced
efficiency of psbL editing, but not of the other four
sites, in the transplastomic lines indicates
depletion of psbL-specific editing factor(s). This
finding implicates the involvement of site-specific
factors in editing of plastid mRNAs in higher plants.
In addition to psbL, editing was shown to create
the AUG translation initiation colon for ndhD in
tobacco (Neckermann et al., 1994). To test whether
editing of initiation colons involves a common
depletable traps-factor, a chimeric gene containing
the ndhD editing site was expressed in tobacco
plastids. The data show that, as for psbL, editing
of the ndhD site requires a depletable ~-rans-factor.
However, this traps-factor is distinct from that
required for psbL editing.
In maize plastids, the translation initiation
colon of rpl2 is created by editing (Hock et al.,
1991). To test, whether the ACG colon in the maize
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18
rpl2 context is edited in tobacco plastids, a
Orpl2/kan gene was constructed by translationally
fusing the N-terminal segment of rpl2 with the kan
coding region. The chimeric mRNA is not edited in
tobacco, but provides a useful selectable marker in
cereals such as maize and rice.
The following examples are provided to merely
illustrate typical protocols for carrying out the
instant invention. They are not intended to limit
the scope of the invention in any way.
EXAMPLE I
EDITING-BASED Ekan and EaaDA SELECTABLE MARKER GENES
A. Construction of the chimeric Genes
The psbL gene encodes a peptide of photosystem
II and is part of the psbE operon (Carillo et al.,
1986; Figure 1 above). A 98-nucleotide fragment
spanning the psbL editing site, ~psbF/OpsbL, was
cloned upstream of the spectinomycin resistance gene
(aadA) coding sequence such that the N-terminus of
psbL was translationally fused with aadA. The
dpsbF/~psbL fragment contains 40 nucleotides of the
psbF C-terminus, 22 nucleotides of the intergenic
region between psbF and psbL and 36 nucleotides of
the psbL N-terminus. The construct was cloned in the
Prrn/Trpsl6 plastid expression cassette (Figure 2A).
Prrn contains the plastid rRNA operon promoter, a
ribosome binding site and a translational initiation
colon (ATG). In the chimeric construct, the
truncated psbF coding region forms an open reading
frame with the Prrn initiation colon (ATG), whereas ,
the translation of the EaadA reading frame (psbL-aadA
fusion peptide) is dependent on the creation of a
translation initiation colon (AUG from ACG) by
editing the psbL site. Note that the two coding
CA 02257867 2002-05-24
19
regions are in different reading frames in the EaadA
mRNA. See Figure 2A.
The Ekan gene was obtained by translationally
fusing psbL with kan, a kanamycin resistance gene
encoding neomycin phosphotransferase using the same
~psbF/apsbL fragment, shown in Figure 2B. Ekan is
similar to the EaadA gene, except that it has 39
nucleotides instead of 36 nucleotides of the psbL
N-terminus. A detailed technical description of the
20 gene construction is set forth below.
The Ekan gene, shown in Figure 2B, in plasmid
pJLMlB was constructed in a pBluesc:ript~KS+ plasmid
(Stratagene). The Ekan coding region in pJLMl8 is
expressed in the Prrn/Trpsl6 cassette. The Prrn
5'-regulatory region consists of the plastid rRNA
operon promoter and a ribosome binding site and is on
an EcoRI/Ncol fragment. Prrn derives from plasmid
pZS195, the progenitor of plasmid pZS197 (Swab and
Maliga, 1993) in which the translational initiation
codon (ATG) is included in the NcoI site. The NcoI
site of Prrn was ligated to the BspHI site of a
BspHI/ XbaI fragment; the NcoI/BspHT fusion
eliminated both restriction sites. The BspHI/XbaI
oligonucleotide was obtained by annealing the
overlapping 5'CATTCATGACTTTGGGATCAATATCAGCATATGCA
GTTCATCCAACGATAAACTTAATCCGAATTATAGAGC-3' and
5'CGGTCTGAATTCAATTCAACATTTTGTTCGTTCGGGTTTGATTGTGTCGTA
GCTCTATAATTCGGATTAAG-3' single-stranded
oligonucleotides and extension with the Klenow
fragment of DNA polymerase I. The BspHI/Xbal
fragment contains the sequence framed in Figure 2B,
including the ~psbF/~psbL sequence encoding the
C-terminal end of psbF, the intergenic region and the
N-terminal portion of psbL. As the result of the
NcoI/BspHI fusion, the C-terminal end of psbF is
translated from the Prrn translational initiation
codon (ATG). To translationally fuse the 14
*Trade-mark
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N-terminal colons of psbL with the kan coding region,
the XbaI single-stranded overhang of the BspHI/XbaI
fragment and the single-stranded overhang of the Ncol
site of kan (including the translational initiation
5 colon) was removed by mung bean nuclease treatment,
and subsequently ligated. The kan coding region
derives from plasmid pTNH4 as an NcoI/Xbal-fragment
(Carrer et al., 1993). The T.rpsl6 fragment is
contained within an XbaI/ HindIII fragment, and was
10 linked to the Ekan coding region via the Xbal site.
The Trpsl6 fragment contains the ~pslG gene
3'-regulatory region between nucleotides 5,087 to
4,939 in the ptDNA {Shinozaki et al., 2986). The
XbaI-site at the 5'-end of the fragment was created
15 by oligonucleotide -directed mutagenesis; the 3'-end
of the fragment was excised from the plastid genome
at an EcoRI-site at nucleotide position 4,938. (Staub
and Maliga, 1994). The EcoRI-site was subsequently
converted to a HindIII-site by linker-ligation. For
20 introduction into the plastid genome, the Ekan
construct was cloned as an EcoRI/HindIII fragment in
the multiple cloning site of plastid vector pPRV111B
(Zoubenko et al., 1994; Gene Bank Accession No.
U12813), which is adjacent to a selectable aadA gene.
The EaadA gene shown in Figure 2A, in plasmid
pJLM20 was constructed in a pBluescript KS+ plasmid
as described for the Ekan gene. The NcvI/XbaI
fragment containing the aadA coding region is derived
from plasmid pHC1 (Carrer et al., 1991) and the aadA
coding region is translationally fused with the 12
N-terminal colons of the tobacco psbL gene. For
introduction into the plastid genome, the EaadA gene ,
was cloned in plastid insertion vector pPRV100B
(Zoubenko et al., 1994, Gene Bank Accession No.
U12811). The pPRV100B vector carries a multiple
cloning site flanked by ptDNA sequences, but no
selectable plastid marker gene.
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While kanamycin, spectinomycin and/or
streptomycin resistance is exemplified herein, the
use of other selectable marker genes is contemplated.
A list of such genes is set forth in Table I below
(Potrykus et al., (1995) in Gene Transfer to Plants,
Springer Verlag.
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TABLE I
Selectable marker genes for plant transformat3.on
Selective agent Marker gene Gene Product
1 Kanamycin, 6418 nptll Neomycin; Phosphotransferase II
O
Gentamycin aacC3 Gentamycin-3-N-acetyltransferase
aacC4
Hygromycin hph, hpt Hygromycin phosphotransferase
Methotrexate dhfr Dihydrofolate reductase
Spectinomycin 16S rDNA 16S rRNA
aadA Aminoglycoside-3'-
adenyltransferase
15 Streptomycin SPT Streptomycin phosphotransferase
16S rRNA
16S rDNA Aminoglycoside-3'-
aadA adenyltransferase
Bleomycin b1e
Phleomycin
Blasticidin bsr Blasticidin S deaminase
Sulfonamide su1 Dihydropteroate synthase
2 Phosphinothricin bar Phosphinothricin acetyltransferase
0
Chlorsulfuron a1s Acetolactate synthase
csr-1
Bromoxynil bxn Bromoxynil nitrilase
Glyphosate EPSPS 5-enolpyruvyl-shikimate-3-
phosphate synthase
2,4-D tfdA 2,4-dichlorophenoxyacetate
monooxygenase
25 Atrazine psbA Q~ protein
2,2-DCPA Dehalogenase
4-methyl-tryptophanetdc Tryptophane decarboxylase
Nitrate NR Nitrate reductase
S-aminoethyl-L- DHPS Dihydropicolinate synthase
3 cysteine
O
lysine/threonine AK Aspartate kinase
aminoethyl-cysteineosc Octopine synthase
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B. Transformation and selection of
antibiotic resistant trans_plastomic lines
The EaadA gene was cloned into the plastid
transformation vector pPRV100B (Zoubenko et al.,
1994) to yield plasmid pHC94 which was introduced
into tobacco chloroplasts by the biolistic process.
The chimeric gene integrated into the plastid genome
via two homologous recombination events in the
trnV-rps7/12 intergenic region. In a sample of 50
bombarded leaves, selection for spectinomycin
resistance resulted in the isolation of 43
spectinomycin resistant clones. Out of these, 34
were confirmed to carry the EaadA gene by DNA gel
blot analysis (data not shown). Expression of
antibiotic resistance indicated editing of the
chimeric EaadA. The efficiency of selection for the
EaadA gene, approximately one plastid transformant
per bombarded leaf sample, was comparable to the
efficiency of selection for an aadA gene whose
expression was independent of editing (Swab and
Maliga, 1993). Three independently transformed
lines, Nt-pHC94-1, Nt-pHC94-10 and Nt-pHC94-11, were
further studied. As direct selection for kanamycin
resistance is inefficient (Carrer et al., 1993), the
Ekan gene was linked to a spectinomycin resistance
gene in transformation vector pPRV111B to yield
plasmid pJLM23. Direct selection of plastid
transformants was attempted after bombardment with
3o pJLM23-plasmid coated tungsten particles. No
kanamycin resistant clones were obtained in a sample
of 200 bombarded leaves {100 each selected on 50
/cg/ml and 100 ~.cg/ml kanamycin sulfate) . However,
transgenic plants containing the Ekan gene were
obtained by selection for the linked
spectinomycin-resistance gene. Three independently
transformed lines, Nt-pJLM23-2, Nt-pJLM~3-14 and
Nt-pJLM23-18, were further studied. Leaf segments
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from each of the clones proliferated on kanamycin
medium (50 /,cg/ml) indicating phenotypic expression of
the Ekan gene. The methods used for plastid
transformation are described in greater detail below.
Tobacco (Nicotiana tabacum cv. Petit Havana)
plants were grown aseptically on agar-solidified
medium containing MS salts (Murashige and Skoog,
1962) and sucrose (30 g/1). Leaves were placed
abaxial side up on RMOP media for bombardment. The
RMOP medium consists of MS salts, N6-benzyladenine
(1 mg/1), 1-naphthaleneacetic acid (0.1 mg/1),
thiamine (1 mg/1), inositol (100 mg/1), agar (6 g/1)
at pH 5.8, and sucrose (30 g/1}. The DNA was
introduced into chloroplasts on the surface of 1 ~m
tungsten particles using the DuPont PDS1000He
Biolistic gun {Maliga, 1995). Spectinomycin
resistant clones were selected on RMOP medium
containing 500 /cg/ml of spectinomycin
dihydrochloride. Resistant shoots were regenerated
on the same selective medium, and rooted on MS agar
medium (Svab and Maliga, 1993). Kanamycin resistant
clones were selected on RMOP medium containing 50 or
100 /~g/ml kanamycin sulfate (Carrer et al., 1993).
C. Editing of EaadA Ekan and psbL transcripts
The phenotypic expression of
antibiotic-resistance by EaadA and Ekan plants
indicated that the chimeric genes were edited since
their translation was made dependent on the editing
of an ACG to an AUG initiation codon. To directly
test for editing of EaadA and Ekan mRNAs, cDNAs were
PCR-amplified with primer 01 within the psbF coding
region and primers 02 and 04 within the EaadA and
Ekan coding sequences, respectively. The position of
primers is shown in Figure 2. The PCR amplification
products are shown in Figure 3A. Direct sequencing
of the PCR products from three independently
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transformed EaadA lines and phosphorimager analysis
indicated that approximately 700 of the EaadA
transcripts are edited. See Figure 3B and Table II.
s TABLE II
Unedited mRNAs (~) in the wild-type
and transQenic leaves
Plant Line Sam 1e sbL EaadA Elzan
10
Nt-wt 1 <0.1
2 0.7
3 0.3
Nt- JLM23-2 1 8.8 30.3
15 Nt- JLM23- 4 1 9.2 28.2
Nt- JLM23- 8 1 10.2 28.4
Nt- HC94-1 1 9.5 28.7
Nt- HC94-10 1 9.0 30.4
Nt- HC94-11 1 10.4 29.9
20
Radioactivity nds in Figure3 corresponding to nucleotides
in
ba
was hosphorimageranalysis. The values were
determined
by
p
normalized loading abeling efficiency against
for and l six
DNA
other same lanes.Percent unedited mRNA =
bands
in
the
2 [corrected signal/(corrected edited + corrected
5 unedited
unedited 100.
signal)]
x
A similar extent of editing was found for the
Ekan mRNAs as shown in Figure 3C and Table II. The
partial editing was not due to the presence of
contaminating DNA in the RNA samples since no
PCR-amplified products were obtained from non-reverse
transcribed DNase I-treated RNA samples. See Figure
3A; lanes 3 and 7. The psbL site in the chimeric
transcripts was only partially (approximately 700)
edited while in leaves of wild-type plants the psbL
mRNA is >99o edited (Kudla et al., 1992; Bock et al.,
1993). Therefore it was of interest to determine
whether or not the editing of the psbL mRNA is
affected in the transgenic plants. The psbL cDNAs
CA 02257867 2002-05-24
26
were PCR-amplified with primers 01. and 06 within the
psbF and psbJ coding regions as shown in Figure 1A,
from wild type and transgenic plants. Direct
sequencing of the PCR products revealed that the
transgenic plants contained approximately 10%
unedited psbL mRNA. This indicates a >10-fold
increase in the level of unedited psbL mRNA in the
transgenic plants. See Figure 4 and Table II.
Artifacts due to DNA contamination of RNA samples
were excluded by the lack of PCR products from
non-reverse transcribed DNase I-treated RNA samples.
See Figure 4A, lanes 3, 7 and 11. Methods utilized
to study the editing in plastid mRNAs are set forth
below.
Total cellular DNA was isolated according to
Mettler (1987). Total cellular RNA was extracted
using TRI2o1*(Gibco BRL). Reverse trar.~cription of
proteinase K- and DNAse I- treated RNA samples were
carried out as described by Kudla et a1.(1992). DNA
and cDNA were amplified by PCR according to standard
protocols: 1 min at 92°C, 2 min at 55°C, 1.5 min at
72°C, 30 cycles.
The PCR amplification products were separated in
1.5% agarose gels and purified using the Geneclean*II
kit (BIO 101 Inc.). Direct sequencing of DNA
was performed as described (Bachmann et al:, 1990)
using the Sequenase~kit (USB) and the detergent
Nonidet~ P-40.
The following is a list of primers used for PCR.
O1 5' -CAATATCAGCAATGCAGTTCATCC-- 3'
02 5' -CCAAGCGATCTTCTTCTTGTCCAA-- 3'
03 5' -GCGCTCGATGACGCCAAC- 3'
04 5' -CACGACGAGATCCTCGCCG- 3'
05 5' -GAATAGCCTCTCCACCCA- 3'
06 5' -GGAATCCTTCCAGTAGTATCGGCC- 3'
07 5' -GGAAAATAAAACAGCAAGTAC- 3°
08 5' -CAAATATTGCAAAGTCCCGG- 3°
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09 5' -CCGGATCGCCACCTACAC-
3'
O10 5' -TGGCTATAACAGAGTTTCTC- 3'
011 5' -GGATTTCCAGAAGAAGATGCC- 3'
014 5' -GTTCGTTCGGGTTTGATTGTG- 3'
015 5' -GAACTCAACGGGCCCTTCCCC- 3'
016 5' -GGAGGGAAGTGGAGTAAATGGC CG-
3'
D. Relative abundance of psbL EaadA and Ekan mRNAs
Accumulation of partially edited p~bL mRNA in
the transgenic lines could be due to its competition
with the chimeric EaadA or Ekan mRNAs for a limiting
common factors) that is required for editing.
Therefore, the relative abundance of the psbL and
chimeric transcripts was determined. It should be
noted that both the polycistronic psbE (Carillo et
al., 1986) and the EaadA and Ekan mRNAs are
approximately 1.1-kb in size. To quantify the
accumulation of these transcripts, differential DNA
probes on Northern blots were utilized. See Figure
5. Probing with the psbJ coding sequence fragment
indicated that the 1.1-kb psbE operon mRNA, which
contains the psbJ and psbL reading fra~t~FS,
accumulates to a similar extent in the wild-type and
transformed plants. See Figure 5B, lower panel. The
014 oligonucleotide probe hybridizes to the mRNA
containing the tlpsbF/~psbL region present in both the
psbE operon and the chimeric EaadA and Ekan
transcripts. The 014 probe detected about 4x more
RNA in the transgenic plants indicating a 1:3 ratio
of the polycistronic psbE to chimeric mRNAs. See
Figure 5B, upper panel. Procedures used for RNA gel
analysis are discussed below.
Total RNA was extracted using TRIzol (Gibco
BRL}. RNA was electrophoresed in formamide-
containing 1o agarose gel and transferred to nylon
membrane (Amersham). Hybridization to 32P-end-labeled
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oligonucleotide probe 014 was carried out in 6 X
SSPE, 0.5% SDS, 10x Dendardt's solution, 100 mg/ml
tRNA, 0.1% Sodium Pyrophosphate at 45°C.
Hybridization to random primed (Boehringer Mannheim)
32P-labeled DNA fragment probes was carried out at
65°C in rapid hybridization buffer (Amersham). RNA
levels in samples that hybridized to the probes were
quantitated by PhosphorImager analysis (Molecular
Dynamics).
E. Editing of other mRNAs is not
affected in the transg~enic plants
Increased demand for psbL editing in the
transgenic plants led to a reduction in its editing
efficiency. Experiments were performed to determine
if editing of other mRNAs is also affected in the
transgenic plants. Two sites were tested in the
rpoB, and two in the ndhB transcripts. See Table
III.
TABLE III
List of tested editing sites
in wild-type and transaenia nlan~~
2 5 Editing Codon no. Codon (aminoacid)
site
Maize Tobacco Unedited Edited
rpoB siteI 156 158 TCG (Ser) TTG (Leu)'
->
rpoB siteII' 182 184 TCA (Ser) TTA (Leu)
--~
ndhB siteI'' 156 156 CCA (Pro) CTA (Leu)
~
ndhB siteII'' 196 196 CAT (His) TAT (Tyr)
-~
'Reference: Zeltz et al., 1993
"Reference: Maier et al., 1992
'In tobacco, a TCA codon is edited to a TTA codon.
35.
The rpoB and ndhB editing sites were originally
reported for maize, and confirmed for tobacco in this
study. Editing sites I and II of rpoB are almost
fully edited in wild-type tobacco, shown in Figure 6,
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as has been observed for maize and barley (Zeltz et
al., 1993). Similarly, sites I and II of the ndhB
transcript are fully edited in wild type tobacco also
shown in Figure 6, as reported for maize (Maier et
al., 1992). The editing efficiency for the same
sites was tested in three lines each of the EaadA-
and Ekan-expressing plants. No significant
difference in the editing efficiency between
wild-type and transformed plants was found for any of
the four sites. Data in Figure 6 are shown for a
Nt-pHC94-1 plant, one of the EaadA-expressing lines.
Lack of change in the editing efficiency at any of
the sites other than psbL indicates that expression
of the chimeric genes specifically compromises the
editing efficiency of the psbL site.
DISCUSSION
The above described examples are the first
demonstration of the editing of chimeric mRNAs in
plastids. Editing of both EaadA and Ekan transcripts
indicates that 98 and 101 nucleotides, respectively,
of the OpsbF/OpsbL fragment are sufficient to direct
editing at the psbL site. Accumulation of EaadA or
Ekan mRNA at levels approximately 3-fold above that
of the psbE polycistronic message containing the psbL
reading frame led to a significant (>10-fold)
increase in the level of unedited psbL transcript.
Increase in the level of unedited psbL mRNA from <1%
to approximately 10o did not have any deleterious
consequence that could have been detected at the
phenotypic level. The chimeric mRNAs were also
partially edited in the transgenic plants.
Partial editing of both psbL and chimeric mRNAs
suggests depletion of a limiting trans-acting
factors) that is required for editing of the shared
site. However, the editing efficiency of four other
sites was unaffected suggesting that the depleted
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factor is specifically required for editing of the
psbL transcript and is not a component of the general
editing machinery. It is therefore conceivable that
each of the editing sites in the chloroplast genome
5 requires some factors for editing that are unique to
them. This conclusion is reinforced by the lack of
any obvious sequence motif common to the 98
nucleotide OpsbF/~psbL fragment and sequences
surrounding the other four tested editing sites.
10 Therefore, it appears likely that the editing of
these sites is directed by sequences and factors that
are unique to each.
As an alternative to depletion of a
site-specific factor, existence of "strong" and
15 "weak" editing sites was also considered.
Accordingly, the psbL site would be weak and its
editing frequency would be lowered by the presence of
excess chimeric RNA competing for a limiting but
common editing factor, whereas the others would be
20 strong sites that remain unaffected. This
explanation is considered unlikely based on other
data in the literature which are consistent with the
existence of site-specific editing factors in
plastids. The psbF mRNA is edited in spinach
25 plastids by a C to U conversion, changing a serine to
a conserved phenylalanine colon. In tobacco at this
position a phenylalanine colon is already present at
the DNA level. When the tobacco psbF gene was
modified to match the spinach sequence, the
30 heterologous editing site was unedited, although the
adjacent psbL site is edited in both species (Bock et
al., 1994). It appears therefore that tobacco lacks
the capacity to edit the spinach psbF mRNA while
maintaining the capacity to edit the psbL site which
is common to both species. Another case consistent
with site-specific editing is site IV of the rpoB
mRNA which is edited in maize but not in barley,
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although the sequences surrounding the site are
highly conserved. Interestingly, the editing of
three other sites in the same transcript is conserved
between the two species (Zeltz et al., 1993). These
observations suggest that the editing capacity of an
individual site may be lacking without affecting the
editing capacity of other sites, supporting
site-specific editing in plastids.
1O EXAMPLE II
EDITING BASED ~psbL/kan AND
OndhD/ltan SELECTABLE MARKER GENES
In plastids, editing of an ACG codon to an AUG
codon creates the translation initiation codon for
25 the psbl and ndhD transcripts in tobacco. To
identify the RNA segment required for psbL editing,
chimeric kanamycin resistance genes were constructed
containing psbL deletion derivatives, and tested in
vivo for editing in transgenic plants. The data
20 demonstrate that a 22 nucleotide segment is
sufficient to direct efficient psbL editing,
including 16 nucleotides upstream and 5 nucleotides
downstream of the editing site. Mutation of the A
nucleotide to a C upstream of the editing site
25 completely abolished editing, while mutation of the
downstream G to a C only reduced the editing
efficiency. Out of the 22 pt editing target
sequence, the 15 upstream nucleotides were found to
compete with the endogenous psbL transcript for a
30 depletable traps-factor. To test whether editing of
initiation codons involves a common traps-factor, a
chimeric gene containing the ndhD editing site was
expressed in tobacco plastids. As for psbL, editing
of the ndhD site requires a depletable traps-factor.
35 However, the ndhD traps-factor is distinct from that
required for psbL editing. Distinct cis-sequences
and traps-factor requirements for the psbL and ndhD
editing sites indicates an individual recognition
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mechanism for the editing of plastid initiation
codons.
A. Defining the cis-sequences directing psbL editing
As mentioned previously, the psbL gene is part
of the psbE operon which contains the psbE, psbF,
psbL and psbJ reading frames (Carilla et al., 1986).
In the earlier examples (Chaudhuri et al., 1995) the
editing of the psbL translation initiation site in a
chimeric mRNA containing a 98 nt OpsbF/~lpsbL fragment
was described {-63/+34 in plasmid pSC2, Figure 7A).
In the chimeric construct of Figure 7A, the first
open reading frame is a truncated psbF (OpsbF) gene
containing 40 nt of the C-terminus. The second open
reading frame contained 36 nt of the N-terminus of
psbL (~psbL) translationally fused with the bacterial
kanamycin resistance (kan) gene to yield the
OpsbL/kan fusion protein. The two open reading
frames are separated by 22 nt of intergenic region.
See Figure 7A.
To identify the sequences required for psbL
editing, deletion derivatives of the 98 nt
OpsbF/OpsbL fragment were tested for editing in vivo.
As before, the psbL deletion derivatives were fused
N-terminally to bacterial kanamycin resistance gene
(kan), and cloned in the plastid Prrn/Trpsl6
expression cassette to create chimeric genes. See
Figure 7A. Thus, for all the constructs, translation
of OpsbL/kan was made dependent on editing of the
psbL ACG codon to AUG codon. Editing therefore could
be tested by the kanamycin resistance phenotype. The
only exception is the chimeric gene containing -2/+34
fragment (in plasmid pSClO) where the initiation
codon for the translation of dpsbL/kan reading frame
was provided by Prrn. The psbL deletion derivatives
were introduced into the tobacco plastid genome by
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33
linkage to a selectable spectinamcyin resistance gene
(Chaudhuri et al., 1995).
The upstream deletion series included constructs
with 5'-ends at positions -63, -51, -39, -27, -16,
-20 and -2 nucleotides relative to the editing site
(position 0). The downstream deletion series
included constructs with 3'-ends at positions +34,
+22, +10, +5 and +1 nucleotides relative to the
editing site. The editing efficiency of the chimeric
mRNAs was determined by direct sequencing and
phosphoimager analysis of PCR-amplified cDNAs.
Editing in the deletion derivatives was maintained as
long as the constructs contained 16 nt of upstream
and 5 nt of the downstream sequence relative to the
editing site as shown in Figures 7A and B.
Interestingly, in the deletion series, the % of the
chimeric mRNA that is edited (editing efficiency) was
either similar to that of the full size 98 nt
OpsbF/OpsbL fragment (about 50%-700), or barely
detectable (--O%). Expression of kanamycin resistance
was also a reliable qualitative marker of editing in
all transformants in which translation of the
chimeric mRNA was dependent on editing. See Figure
7A. The exception were plants obtained by
transformation with plasmid pSClO in which kanamycin
resistance is expressed from the translation
initiation codon contained in the Prrn promoter
fragment. Construction of the deletion derivatives is
set forth below.
The psbL deletion derivatives and the ndhD gene
fragment were generated by PCR amplification with 5'
primers carrying Ncol restriction site and 3' primers
carrying NheI restriction site using total cellular
DNA from tobacco (cv. Petit Havana). The following
primer pairs were used: plasmid pSC2, 023 and 029;
plasmid pSC3, 023 and 030; plasmid pSC4, 023 and 031;
plasmid pSC5, 023 and 032; plasmid pSC6, 024 and 029;
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plasmid pSC7, 025 and 029; plasmid pSC8, 026 and 029;
plasmid pSC9, 027 and 029; plasmid pSClO, 028 and
029; plasmid pSCl8, 027 and 031; plasmid pSCl9, 027
and 034; plasmid pSC20, 033 and 031; pSC23, 037 and
038. The PCR products were digested with NcoI and
NheI restriction enzymes. '
To introduce suitable restriction sites at the
5'-end of the kan coding region, kan was PCR
amplified from pTNH32 (Carrer et al., 1993) using 5'
primer (021) carrying NcoI and Nhe2 restriction sites
in tandem and 3' primer (022) carrying XbaI
restriction site. The PCR product was cloned in
NcoI/XbaI digested pUC120 to generate plasmid pSCl.
The chimeric genes were constructed by
N-terminal fusion of PCR amplified sequences from
tobacco psbL and ndhD genes (NcoI/NheI fragments) to
bacterial kan gene lacking the initiation codon
(NheI/XbaI fragments). The chimeric genes were then
cloned in NcoI/XbaI digested plasmid pLAA24A
(Zoubenko et al., 1994). Plasmid pLAA24 is a
derivative of plastid transformation vector pPRV111A,
(Gene Bank Accession No. U12822) which has a
selectable spectinomycin resistance gene, and a uidA
reporter gene in the Prrn/Trpsl6 expression cassette
(zoubenko et al., 1994). The Prrn 5'-regulatory
region consists of the plastid rRNA operon promoter
and a ribosome binding site and is on an SacI/NcoI
fragment. The Trpsl6 fragment includes the rpsl6
gene 3'-regulatory region between nucleotides 5,087
to 4,939 in the ptDNA (Shinozaki et al., 1986) and is
contained within an XbaI/HindII2 fragment. Digestion
of plasmid pLAA24A with NcoI/XbaI restriction enzymes _
removes the uidA coding region from the expression
cassette, which is then replaced with the chimeric
constructs, also an NcoI/XbaI fragment.
To construct the chimeric genes of the
subsequent examples, the following procedures were
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used. Plastid transformation and plant regeneration
were performed as described in Example I. PCR
amplification and DNA sequencing were also performed
~ as described in Example I above. The sequencing gels
5 were subjected to phosphoimager analysis (Molecular
~ Dynamics) for quantitation of editing efficiency.
Radioactivity in bands corresponding to nucleotides
was determined. The values were normalized for
sample loading and labeling efficiency against other
10 bands in the same lanes. mRNA editing efficiency
- [corrected edited signal/(corrected edited +
corrected unedited signal)] X 100. The primers used
were as follows:
01: 5'-CAATATCAGCAATGCAGTTCATCC-3'
15 04: 5'-CACGACGAGATCCTCGCCG-3'
05: 5'-GAATAGCCTCTCCACCCA-3'
06: 5'-GGAATCCTTCCAGTAGTATCGGCC-3'
07: 5'-GGAAAATAAAACAGCAAGTAC-3'
017: 5'-AATTCGAAGCGCTTGGATACAGTTGTAGGGA-3'
20 018: 5'-GTAAGAGATGTGAATCCGCCTGT-3'
019: 5'-GCATAAGTCGTTAGAAGGAG-3'
020: 5'-GAAGAAAGAAAATTAAGGAACC-3'
021: 5'-CATGCCATGGCTAGCATTGAACAAGATGGATTGCACG-3'
022: 5'-GTACTCTAGACCCGCTCAGAAGAACTCG-3'
25 023: 5'-CTAGCCATGGCTTTGGGATCAATATCAGCAATG-3'
024: 5'-CTAGCCATGGCATCAGCAATGCAGTTCATCC-3'
025: 5'-CTAGCCATGGCGTTCATCCAACGATAAACTTAA-3'
026: 5'-CTAGCCATGGCATAAACTTAATCCGAA.TTATAGAG-3'
027: 5'-CTAGCCATGGCC.GAATTATAGAGCTACGACAC-3'
30 028: 5'-CTAGCCATGGCTACGACACAATCAAACCCGA-3'
029: 5'-CTAGCTAGCTTCAACATTTTGTTCGTTCGG-3'
030: 5'-CTAGCTAGCTTCGTTCGGGTTTGATTGTG-3'
031: 5'-CTAGCTAGCTGATTGTGTCGTAGCTCTATA-3'
032: 5'-CTAGCTAGCCGTAGCTCTATAATTCGGATT-3'
35 033: 5'-CTAGCCATGGTATAGAGCTACGACAC-3'
034: 5'-CTAGCTAGCAAGTGTCGTAGCTCTATA-3'
035: 5'-AATTATAGAGCTCCGACACAATC-3'
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36
036: 5'-AATTATAGAGCTACCACACAATC-3'
037: 5'-CTAGCCATGGTATTTTGAGCACGGGTTTTTCTGGTCC-3'
038: 5'-CTAGCTAGCTGGAAAAACTACAATTATTGTTAACC-3'
B. Mutation of the nucleotides
flanking the psbL editing site
The edited ACG colon to CCG and ACC in the
efficiently edited 98 nt OpsbF/~psbL fragment were
altered to address the following issues: (1) Whether
the flanking nucleotides are critical for editing.
(2) Whether the fidelity of editing the correct C is
maintained when one of the flanking nucleotides is
changed to a C. (3) Whether translation initiation at
this site is required for editing, since changing the
ACG colon to CCG and ACC would eliminate the
possibility of translation initiation at the edited
colon.
Mutation of the upstream nucleotide (ACG to CCG;
NtpSCl4 line) resulted in the loss of editing (-O~).
See Figure 8. Mutation of the downstream nucleotide
(ACG to ACC; Nt-pSCl5 line) allowed editing at the
correct C, but at a significantly reduced efficiency,
~20%. See Figure 8. The mutational analysis
therefore indicated that the A residue directly
upstream of the edited C is appears to be essential
for editing while mutation of the downstream G
residue to C is compatible with editing but is
required for optimal efficiency. In addition,
editing of the correct C in the mutated colon ACC
points to a high fidelity mechanism of the editing
apparatus in the choice of the editing site.
Furthermore, editing of the ACC colon suggests that
translation initiation at this colon is not required
for editing. Construction of the chimeric genes and
introduction into plants was carried out as described
in section A for the OpsbL/kan derivatives.
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37
The psbL derivatives with a point mutation were
obtained by the megaprimer method of PCR (Sarkar and
Sommer, 1990) using plasmid pSC2 as the template.
These were also designed as NcoI and NheI fragments.
The primers used were the following: plasmid pSCl4,
step I, 035 and 029, step II, 023; plasmid pSClS,
step I, 036 and 029, step II, 023.
C. Identification of psbL mRNA sequences which
interact with a psbL-specific editing factor
_( bsbL-SEF) .
In previous examples it has been shown that the
editing efficiency of the endogenous psbL transcript
is reduced in plastids expressing the chimeric psbL
mRNA. Reduced editing efficiency was due to
competition of the 98 nt OpsbF/OpsbL fragment with
the endogenous psbL mRNA for a site-specific editing
factor (psbL-SEF) present in limiting amounts
(Chaudhuri et al., 1995).
Testing psbL editing efficiency in plastids
expressing the chimeric OpsbF/GlpsbL deletion
derivatives, shown in Figure 7, was used to further
define psbL sequences which interact with psbL-SEF.
Out of the 22 nucleotides minimally required for
editing, only the segment upstream of the editing
site was able to compete with endogenous psbL for
psbL-SEF. The 16 nt psbL-SEF binding site (boxed)
within the 22 nt psbL editing recognition sequence is
shown in Figure 9A. Sequences between nucleotides
-16/-10 are critical for competition since
competition is abolished in plastids containing the
pSC20 construct which lacks this sequence. See
Figure 9B. Interestingly, the plants expressing the
pSCl4 construct with the A to C mutation at position
-1 also maintained competition, although this
mutation completely abolished editing. The psbL
editing efficiency data for the critical constructs
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38
are shown in Figures 9A and 9B. While in the
wildtype plants psbL mRNA is >99% edited, competition
in the transgenic lines lead to accumulation of a
significant amount of unedited psbL transcript.
D. Editing of the ndhD initiation
codon in chimeric mRNA
Sequence analysis of ndhD and the corresponding
ZO cDNA by Neckermann et al (1994) has established that
the ndhD translation initiation codon is created by
editing of an ACG colon to an AUG colon in tobacco,
spinach and snapdragon. The following experiments
were designed to test whether ndhD editing requires a
depletable traps-factor as found for psbL, and
whether this traps-factor is utilized for the editing
of both initiation colon sites. For this purpose,
an 89 nucleotide fragment (-48/+40) spanning the ndhD
editing site was translationally fused with the ken
coding region and cloned in a Prrn/Trpsl6 expression
cassette. See Figure 10 A and B. The chimeric gene
were constructed by N-terminal fusion of PCR
amplified sequences from the tobacco ndhD gene
(NcoI/NheI fragments) to bacterial ken gene lacking
the initiation colon (NheI/XbaI fragments). For a
detailed description of the construction of the
chimeric gene see Example II, section A. The
chimeric gene was introduced into the tobacco plastid
genome by linkage to a spectinomycin resistance gene.
In the chimeric gene, expression of the ~lr2dhD/kan
fusion protein was dependent on the editing of the
ndhD site. To prevent translation from an upstream
AUG, a point mutation was introduced 26 pt upstream
of the editing site changing an A to a C, underlined
in Figure 10 A. ,
Nt-pSC23 plants expressing the OndhD/kan protein
were resistant to kanamycin indicating editing of the
ndhD site. Direct sequencing of PCR amplified
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39
dndhD/kan revealed a very low efficiency (~70) of
editing, shown in Figure 10C. The ndhD transcript in
the wild-type plants is edited at significantly
higher efficiency (-45 ~), which is reduced in the
Nt-pSC23 plants to ~ 20 0. See Figure 10C. The
reduction in the editing efficiency of the endogenous
ndhD transcript in the transgenic plants indicates
that increasing the demand for ndhD editing leads to
the depletion of an editing factor which is present
in limiting amounts. However, the efficiency of
editing of the psbL transcript in the transgenic
Nt-pSC23 plants was comparable to the wild-type
levels, >99 0, shown in Figure 10C. Since psbL
editing in the Nt-pSC23 plants is unaffected, the
depleted editing factor is ndhD-specific, and is not
required for psbL editing.
ndhD editing in plants expressing the
chimeric kan gene fused with the 98 nt GlpsbF/~psbL
fragment, Nt-pSC2, Figure 7, was also examined. In
such plants reduced editing of the endogenous psbL
mRNA due to competition for psbL-SEF has been shown
(Chaudhuri et al., 1995; Figure 10C). However, in
the same plants the endogenous ndhD editing is
unaffected, see Figure 10C, indicating that psbL-SEF
is not involved in editing the ndhD site.
DISCUSSION
The above examples describe the analysis of the
cis-element requirements for mRNA editing in
plastids. The data show that the C to U conversion
in the psbL mRNA is directed by a 22 nucleotide
sequence which encompasses 16 nucleotides upstream
and 5 nucleotides downstream of the edited C at
position 0. The 22 nt sequence is conserved in
tobacco, spinach (Kudla et al., 1992) and bell pepper
(Kuntz et al., 1992), species in which editing of the
psbL translation initiation codon has been reported.
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The role of nucleotides directly flanking the
editing site was tested by mutating them in the 9s nt
~psbF/~psbL fragment which is efficiently edited.
Changing the upstream A at -1 to a C completely
abolished editing of the correct C. However,
changing the G at +1 to a C allowed editing of the
correct C, although at a reduced efficiency. Editing
of the correct C in the mutated ACC colon indicates
the high fidelity of nucleotide selection for
editing. This is consistent with the observation
that specific C nucleotides are edited within
flanking C sequences (Kossel et al., 1993; Maier et
al., 1995). Furthermore, editing of the ACC colon
suggests that translation initiation is not required
for editing to occur, providing direct evidence for
the lack of linkage between translation and editing.
This finding is consistent with mRNA editing in
plastids lacking ribosomes (Zeltz et al., 1993) and
with editing of unspliced plastid mRNAs which are not
translatable (Freyer et al., 1993).
The psbL translation initiation colon is only
one of the approximately 25 editing sites found in
the plastids of higher plants (Maier et al., 1995).
Further studies will be required to determine how
typical is the close proximity of cis-sequences to
wthe editing sites in plastids found for psbL. In
this regard, the ndhD initiation colon appears to be
similar since all information required for editing is
contained in a relatively small (98 nt) RNA segment.
However, editing of sites II and III in the tobacco
ndh8 gene (Maier et al., 1992) requires sequences
further away than 150 nucleotides.
Therefore, localization of editing cis
sequences is not uniform, in line with the proposed
individual recognition mechanism for each of the -25
plastid editing sites.
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41
Individual recognition of the editing sites is
consistent with the finding that site-specific
trans-factors are depleted by over-expression of the
psbL and ndhD target RNAs. While ACG to AUG editing
in both transcripts creates a translation initiation
codon, over-expression of either of the target RNAs
affects the editing efficiency of only the source
mRNA.
It is likely that C to U editing in plastids
involves cytidine deamination, as shown for plant
mitochondria (Yu and Schuster, 1995). Editing
therefore minimally involves either a single
polypeptide containing both a site-specific
recognition domain and a deaminase domain, or a
complex containing at least two components, one
providing site-specific recognition and the other
with cytidine deaminase activity. Such a
mufti-component complex consisting of cytidine
deaminase (APOBEC-1) and auxiliary proteins has been
shown to be involved in C to U editing of the
mammalian nuclear apolipoprotein B mRNA. In addition
to the common occurrence of C to U editing, close
clustering of the cis-sequences around the editing
site is an additional feature shared by the plastid
psbL and the mammalian nuclear apolipoprotein B
editing systems. Editing of apolipoprotein B is
directed by an 11 nucleotide recognition sequence
located four nucleotides downstream of the editing
site. In addition, sequences upstream are required
for efficient editing (reviewed in Innerarity et al.,
1996). However, in contrast to editing of psbL,
recognition specificity of the apolipoprotein B
editing process is relaxed, since cytosines
introduced adjacent to the edited nucleotide may also
be modified (Chen et al., 1990).
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EXAMPLE III
_E_ditina based drpl2/kan selectable marker gene
The chimeric ~z-p12/kan gene was constructed by
N-terminal fusion of PCR amplified sequences from the
maize rpl2 gene (NcoI/NheI fragment; 5'primer:
5'-CTAGCCATGGAAACGAACTAAAGGAGAATAC-3'; 3' primer:
5'-CTAGCTAGCCGGGATAGGTGTTTTGTATAAA-3') to a bacterial
kan gene lacking the initiation codon (NheI/Xbal
to fragments). See Figure 11A and 21B. The chimeric
genes were then cloned in NcoI/XbaI digested plasmid
pLAA24A (Zoubenko et al., 1994), as described for the
construction of ~psbL/kan genes and introduced into
the tobacco plastid genome (Chaudhuri and Maliga,
1996). The chimeric mRNA was transcribed in tobacco
plastids. In tobacco, no editing of the maize rpl2
translation initiation codon was found. Aiso, the
transformed plants were sensitive to kanamycin.
However, editing of this chimeric gene will occur in
rice, maize and other cereals in which the rp.I2
translation initiation codon is created by editing.
EXAMPLE IV
CONVERSION OF INTERNAL EDITING SITES TO
EDITED TRANSLATION INITIATION CODONS
The translation initiation codon is created by
conversion of an ACG codon to an AUG codon in the
psbL, ndhD and rpl2 plastid mRNAs. The psbL (Kudla
et al., 1992) and ndhD (Neckermann et al., 1994)
editing sites are present in a few but not all
dicotyledonous species, whereas the rpl2 site is
edited in most but not all cereals (Hoch et al., ,
1991). The maize rpl2 site is not edited in tobacco
(Chauduri and Maliga, see Example III). Therefore,
the psbL, and ndhD editing sites are useful to create
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43
editing-based marker genes in some divots, and
rpl2-based chimeric genes are useful in most
monocots.
There are many more examples for the editing of
internal codons than for editing of translation
' initiation codons. However, translation initiation
is not required for editing of codons in the psbL
sequence context (Chaudhuri and Maliga, 1996). Based
on these results, internal codons may also serve as
translation initiation codons as long as editing
creates a translatable mRNA. There is a high
frequency of Ser to Phe, Ser to Leu and Pro to Leu
transitions, and a lower extent of Thr to (F)Met
transitions. Given that U and A are relatively
frequent at the first nucleotide position, editing of
UCG codons will be maintained in most editing
contexts even if the first nucleotide is changed to A
to create a codon which may be edited to a
translation initiation codon by C to U conversion.
Good candidates for such mutagenesis are editing
Sites I and II of the rpoB mRNA, which are widely
edited in both divots and monocots (Zeltz et al.,
1993).
RNA sequences required to direct editing may be
contained within a short segment adjacent to the
editing site as in the case of the psbL gene or may
be at a distance as in case of Sites II and III of
the ndhB gene (Chaudhuri and Maliga, 1996). Editing
has been tested in ndhB and rpoB minigenes to
. identify editing sites that are useful for the
construction of chimeric genes.
ndhB and rpoB editing sites have been identified
for which the relevant cis-sequences are within a
short segment. These short gene segments have been
incorporated in chimeric genes, expressed in tobacco
plastids, and tested for editing by direct sequencing
of the PCR-amplified transgene cDNAs. The editing
CA 02257867 2004-05-26
44
sites in the source genes are listed in Table IV. The
map of the ndhB and rpoB minigene derivatives is
shown in Figure 12.
TABLE IV
RNA editing in ndhB and rpoB minigenes.
Editing Codon
(amino
acid)
site Codon Unedited / Edited
No Reference
1 0 _____ ___________________________________________________________
rpoB siteI 158 "TCG (Ser)to TTG(Leu) Zeltz etal., 1993
rpoB siteII 184 TCA (Ser)to TTA(Leu) Zeltz etal., 1993
ndhe siteI 156 CCA (Pro)to CTA(Leu) Maier etal., 1992
ndhB siteII 196 CAT (His)to TAT(Tyr) Maier etal., 1992
1 5 ndhB siteIII204 TCA (Ser)to TTA(Leu) Maier etal., 1992
ndhB siteIV 246 CCA (Pro)to CTA(Leu) Maier etal., 1992
ndhB siteX 249 TCT (Ser)to TTT(Phe)
2 0 'In tobacco, a TCA codon is edited to a TTA codon (Chaudhuri et
al., 1995).
The ndhB minigene contains an ndhB fragment
25 which is 369 nucleotide in size (between nucleotides
143,174 and 144,042 of the plastid genome, Shinozaki
et al., 1986). It contains some of the first exonic
sequence including five editing sites, named as sites
I, II, III, IV and X. Sites I, II, III and IV are
30 edited in maize, rice and tobacco (Maier et al.,
1992). Therefore, marker genes based on the editing
of these sites will be useful in a broad range of
crops, including monocots and dicots. Site X is
' -edited in tobacco only, therefore this site is less useful
35 for the construction of chimeric marker genes.
In the ndhB minigene, the truncated coding
region is expressed in the original reading frame, in
the Prrn-Trpsl6 cassette. This was achieved by
40 introducing an NcoI site at the 5~-end of the
truncated reading frame, which includes the
translation initiation codon (CCATGG) from which the
minigene RNA can be translated from translation
signals contained in the cassette. The minigene
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contains the DNA sequence ATGGCAGCTACT downstream of
the translation initiation codon; nucleotide C at
position five corresponds to nucleotide 143,674 in
' the plastid genome. In addition, during PCR
5 amplification, an in-frame stop codon was introduced
' at the 3'-end of the truncated coding region. (5' PCR
primer: 5'-CTAGCCATGGCAGCTACTCTAGGGGGAATG-3'; 3' PCR
primer: 5'-CTAGTCTAGACGTATACGTCAGGAGTCCA-3'
The
minigene was physically linked to a selectable
~0 spectinomcyin resistance (aadA) gene in a suitable
plastid targeting vector and the vector DNA was
introduced into tobacco leaf chloroplasts by the
biolistic process. Transplastomes with the
integrated, linked transgenes were selectively
15 amplified by incubating the bombarded leaf segments
on a spectinomycin medium, on which transgenic shoots
were directly regenerated. The protocols for plastid
transformation have been described in Svab and
Maliga, 1993 and Zoubenko et al., 1994.
20 Out of the five sites, Sites I, IV and X were
highly edited in the minigene. This finding
indicates, that the cis sequences required for
editing are located relatively close to the editing
sites, as was shown for the psbL translation
25 initiation codon (Chaudhuri and Maliga, 1996).
Furthermore, cis sequences for Sites I and IV are
suitable for inclusion in marker genes with utility
in both dicots and monocots, since the capacity for
editing is present in these widely divergent
30 taxonomic groups. Interestingly, ndhB Sites II and
III were not edited in the minigene, indicating that
the cis sequences required for editing are further
away than +/- 150 nucleotides from the editing site.
Therefore, cis sequences required for editing are not
35 uniformly positioned relative to the editing site.
The rpoB minigene contains a 281 nucleotide
fragment of the rpoB gene, encoding the RNA
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46
polymerase ~i-subunit. The fragment contains two
editing sites (I, II, see Table IV and Fig. 12; based
on Zeltz et al., 1993). Both editing sites are
present in maize, rice, barley, spinach and tobacco
(Maier et al., 1992). The rpoB minigene was
constructed and introduced into plastids as described
for the ndhB minigene. The minigene contains the DNA
sequence ATGGTCCCGGT downstream of the translation
initiation codon; nucleotide G at position four
corresponds to the complement of nucleotide 27120 in
the plastid genome (Zhinozaki et al., 1986). The
rpoB fragment for the minigene construction was
obtained by PCR amplification (5' PCR primers:
5~-CTAGCCATGGGTCCCGGTATTTATTACCG-3T;
3' PCR primer:
5'-CTAGGTCGACTTAGGCATTTTCTTTTGACCCAAT-3'). Transgenic
plants representing several independently transformed
lines were obtained and assayed for editing. Complete
editing of both of the sites was found in the
minigenes by sequencing PCR-amplified cDNAs. Given
the presence of these sites in both monocots and
dicots, marker genes based on the editing of either
of these sites could be used in a wide variety of
crops.
EXAMPLE V
RNA EDITING FOR TISSUE-SPECIFIC
REGULATION OF FOREIGN GENE EXPRESSION
35
RNA editing in plastids was discussed assuming
that editing is constitutive, and facilitates _
expression of marker genes in all tissue types. It is
known, however, that environmental and developmental
conditions significantly affect editing efficiency
(Buck et al., 2993; Hirose et al., 1996). Tissue
specific differences in editing efficiency facilitate
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47
the design of chimeric genes the translation of which
is dependent on tissue type due to tissue-specific
conversion of ACG colons to a translation initiation
colon. Such chimeric genes are useful when
accumulation of an economically useful protein, such
as an insecticidal endotoxin is desired only in
specific tissue types, such as leaves, root hairs,
root cortex or epidermis cells.
Alternatively, desired tissue-specific
expression of economically useful genes may be
obtained when editing tissue specifically creates a
translation termination (stop) colon. Formation of
stop colons by editing may be readily obtained by
engineering in plastids. For example, a stop colon in
plastids is created when changing the reading frame
of the psbL editing site. Normally, the psbL
translation initiation colon is created by C to U
conversion in the ALGA sequence. Moving the reading
frame by one nucleotide, editing creates the TGA
stop colon. Editing of the first C nucleotide of a
colon is also known, such as Site II of the ndhB
transcript (Maier et al., 1992). Therefore, C to U
editing of the CAA colon will create the TAA
translation termination colon.
Most plastid genes are organized in
polycistronic transcription units. Therefore,
polycistronic transcription units may be built for
simultaneous expression of multiple proteins. An
example for a dicistronic transcription unit, form
which two proteins are simultaneously expressed was
obtained by engineering of the plastid genome (Staub
and Maliga, 1995). Tissue-specific expression of such
polycistronic mRNAs may be obtained by making the
translation dependent on RNA editing, either through
creation of a translation initiation colon, or by
terminating translation.
CA 02257867 2004-05-26
48
REFERENCES
1. Araya, A., Domec, C., Begu, D., and Litvak, J.
(1992) an in vitro system for editing of ATP synthase
subunit mRNA using wheat mitochondrial extracts.
Proc. Natl. Acad. Sci. USA, 89, 1040-1044.
2. Benne, R. (1994) RNA editing in trypanosomes.
Eur. J. Biochem. 221, 9-23.
3. Bachmann,B., Lueke,W. and Hunsmann,G. (1990)
Improvement of PCR amplified DNA sequencing with the
aid of detergents. Nucleic Acids Res., 18, 1309.
4. Bock,R " Hagemann,R., Kossel,H and Kudla,J
(1993) Tissue- and stage-specific modulation of RNA
editing of the psbF and psbL transcript from spinach
plastids - a new regulatory mechanism? Mol. Gen.
Genet., 240, 238-244.
30
5. Bock,R., and Maliga,P. (1995) In vivo testing of
a tobacco plastid DNA segment for guide RNA functio_n___
in psbL editing. Mol. Gen. Genet., 247(4):439-43.
6. Bock,R., Kossel,H. and Maliga,P. (1994)
Introduction of a heterologous editing site into the
tobacco plastid genome: the lack of RNA editing leads
to a mutant phenotype. EMBO J., 13, 4623-4628.
7. Bonnard, G., Gualberto, J.M. (1992) RNA editing
in plant mitochondria. Critical Reviews in Plant
Sciences, 10, 503-524.
8. Carillo,N., Seyer,P., Tyagi,A. and Herrmann,R.G.
(1986) Cytochrome b-559 genes from Oenothera hookeri
and Nicotiana tobacco show a~remarkably high degree
~f conservation as compared to spinach. The enigma of
~cytochrome b-559: Highly conserved genes and proteins
but no known function. Curr. Genet., l0, 619-624.
9. Carrer,H., Hockenberry,T.N., Svab,Z. and
Maliga,P. (1993) Kanamycin resistance as a selectable
marker for plastid transformation in tobacco. Mol.
Gen. Genet., 241, 49-56.
10. Carrer,H., Staub, J.M. and Maliga,P. (1991)
Gentamicin resistance in Nicotiana conferred by
AAC(3)-I, a narrow substrate specificity
acetyltransferase. Plant Mol. Biol., 17, 301-303.
11. Chan,L. (1993) RNA editing: exploring one made
with apolipoprotein B mRNA. BioEssays 15, 33-41.
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49
12. Chaudhuri, S. and Maliga, P. (1996) Sequences
directing to C to U editing of the plastid psbL mRNA
are located within a 22 nucleotide segment spanning
the editing site. E'MBO J., accepted, pending on
revisions.
13. Chaudhuri, S., Carrer, H. and Maliga, P. (1995)
Site-specific factor involved in the editing of the
psbL mRNA in tobacco plastids. EMBO J. 14, 2951-
2957.
14. Cornelissen M, Vandewiele M. (1989) Nuclear
transcriptional activity of the tobacco plastid psbA
promoter. Nucleic Acids Res. 17:19-29.
15. Freyer, R., Hock, B., Neckermann, K., Maier,
R.M. and Kossel, H. (1993) RNA editing in maize
chloroplasts is a processing step independent of
splicing and cleavage to monocistronic mRNAs. The
Plant J., 4, 621-629.
16. Gray,M.W. and Covello,P.S. (1993) RNA editing in
plant mitochondria and chloroplasts. FASE'B J., 7,
64-71.
17. Hanson, M.R., Sutton, C.A. and Lu,B. (1995)
Plant organelle gene expression: altered by RNA
editing. Trends in plant Sciences, 1, 57-64.
18. Higuchi,M., Single,F.N., Kohler,M., Sommer,B.,
Sprengel,R. and Seeburg, P.H. (1993) RNA editing of
AMPA receptor subunit GluR-B: a base-paired
intron-exon structure determines position and
efficiency. Cell, 75, 1361-1370.
19. Hirose, T, Fan, H, Suzuki, Y.Y., Wakasugi, T.,
Tsudzuki, T., Kossel, H. and Sugiura, M. (1996)
Occurrence of silent RNA editing in chloroplasts:
its species specificity and the influence of
environmental and developmental conditions. Plant
Mol. Biol. 30:667-672.
20. Hock, B., Maier, R.M., Appel, K., Igloi, G.L.
and Kossel, H. (1991) Editing of a chloroplast mRNA
by creation of an initiation codon. Nature 353: 178-
180.
21. Hodges,P. and Scott,J. (1993) Editing of
mammalian apolipoprotein B mRNA by site-specific RNA
deamination. In Benne,R. (ed.), RNA editing. The
alteration of protein coding sequences of RNA. Ellis
Horwood, New York, NY., pp. 125-151.
22. Innerarity, T.L., Boren, J., Yamanaka, S. and
Olofsson, S.O. (1996) Biosynthesis of apolipoprotein
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B48-containing lipoptotiens. J. Biol. Chem., 271,
2353-2356.
23. Koncz, C.S., Martini, N., Mayerhofer, R., Konca-
5 Kalman, Z.S., Korber H., Redei, G.Y., Schell, J.,
(1989) High-frequence T-DNA-mediated gene tagging in
plants. Proc. Nat1 Acad. Sci. USA 86:8467-8471.
24. Kossel,H., Hoch,B., Maier,R.M., Igloi,G.L.,
10 Kudla,J., Zeltz,P., Freyer,R., Neckermann,K. and
Ruf,S. (1993) RNA editing in chloroplasts. In Kueck,
U. and Brennicke,A. (eds.), Plant Mitochondria. VCH
Publishers, Weinheim, Germany, pp. 93-102.
15 25. Kudla,J., Igloi,G.L., Metzlaff,M., Hagemann,R.
and Kossel, H. (1992) RNA editing in tobacco
chloroplasts leads to the formation of a translatable
psbL messenger RNA by a C-to-U substitution within
the initiation colon. EMBO J., 11, 1099-1103.
26. Kuntz, M., Camara, B., Weil, J.H. and Schantz,
R. (1992) the psbL gene from bell pepper (Capsicum
annuum): plastid RNA editing occurs in non-
photosynthetic chromoplasts. Plant. Mol. Biol. 20,
1185-1188.
27. Maier,R.M., Neckermann,K., Hoch,B.,
Akhmedov,N.B, and Kossel,H (1992) Identification of
editing positions in the ndhB transcript from maize
chloroplasts reveals sequence similarities between
editing sites of chloroplasts and plant mitochondria.
Nucl. Acids Res., 20, 6189-6194.
28. Maier, R.M., Neckermann, K., Igloi, G.L. and
Kossel, H. (1995) Complete sequence of the maize
chloroplast genome: gene content, hotspots of
divergence and fine tuning of genetic information by
transcript editing. J. Mol. Biol. 251, 614-628.
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32. Murashige,T. and Skoog,F. (1962) A revised
medium for rapid growth and bioassays with tobacco
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H., and Maier, R.M. (1994) The role of RNA editing
in convervation of start codons in chloroplast
genomes. Gene, 146, 177-182.
34. Sarkar, G. and Sommer, S.S. (1990) The
"megaprimer" method of site-directed mutagensis.
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35. Schuster,W. and Brennicke,A. (1994) The plant
mitochondria) genome: physical structure, information
content, RNA editing, and gene migration. Annu. Rev.
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36. Schuster, W. and Brennicke, A. (1994) The plant
mitochondria) genome: physical structure, information
content, RNA editing, and gene migration. Annu. Rev.
Plant Physiol. Plant Mol. Biol., 45, 61-78.
37. Scott, J. (1995) A place in the world for RNA
editing. Cell, 81, 833-836.
38. Shinozaki,K. et al. (2986) The complete
nucleotide sequence of the tobacco chloroplast
genome: Its gene organization and gene expression.
EMBO J., 5, 2043-2049.
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editing in Leishmania mitochondria. In Benne,R.
(ed.), RNA editing. The alteration of protein coding
sequences of RNA. Ellis Horwood, New York, NY., pp.
53-85.
40. Staub,J. and Maliga,P. (1994) Translation of
psbA mRNA is regulated by light via the
5'-untranslated region in tobacco plastids. Plant J.,
6, 547-553.
41. Sutton, C.A., Zoubenko, O.V., Hanson, M.R. and
Maliga, P. (1995) A plant mitochondria) sequence
transcribed in transgenic tobacco chloroplasts is not
edited. Mol. Cell. Biol. 15, 1377-1381.
42. Svab,Z. and Maliga,P (1993) High-frequency
plastid transformation in tobacco by selection for a
chimeric aadA gene. Proc. Nat). Acad. Sci. USA, 90,
9I3-917.
43. Teng,B.B., Burant,C.F., Davidson, N.O. (1993)
Molecular cloning of the apolipoprotein B messenger
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52
44. Yu, W. and Schuster, W. (1995) Evidence for a
site-specific cytidine deamination reaction involved
in C to U editing of plant mitochondria. J. Biol.
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45. Zeltz,P., Hess,W.R., Neckermann,K., Boerner,T.
and Kossel,H. (1993) Editing of the chloroplast rpoB
transcript is independent of chloroplast translation
and shows different patterns in barley and maize.
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22, 3819-3824.
While certain preferred embodiments of the
present invention have been described and
specifically exemplified above, it is not intended
that the invention be limited to such embodiments.
Various modifications may be made thereto without
departing from the scope and spirit of the present
invention, as set forth in the following claims.
CA 02257867 1999-10-04
53
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(1) APPLICANT:
(A) Rutgers, the State University of New Jersey
Maliga, Pal
Carrer, Helaine
Chaudhuri, Sumita
(ii) TITLE OF THE INVENTION: Editing-Based Selectable Plastid
Marker Genes
(iii) NUMBER OF SEQUENCES: 66
(iV) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BORDEN ELLIOT SCOTT & AYLEN
(B) STREET: 60 QUEEN STREET
(C) CITY: OTTAWA
(D) PROVINCE: ONTARIO
(E) COUNTRY CANADA
(F)POSTAL CODE: K1P 5Y7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatable
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: FastSEQ for Windows Version 3.0
(vi) CURRENT APPLICATION DATA:
(A) PATENT APPLICATION: CA 2,257,867
(b) FILING DATE: 1998-12-10
(vii) PRIOR PATENT INFORMATION:
(A) EARLIER PATENT APPLICATION: PCT/US97/10318
(B) EARLIER APPLICATION FILING DATE: 1997-06-13
(A) EARLIER PATENT APPLICATION: 60/019,741
(B) EARLIER APPLICATION FILING DATE: 1996-06-14
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: JOACHIM T. FRITZ
(B) REGISTRATION NUMBER: 4173
(C) REFERENCE/DOCKET NUMBER: PAT 43724W-1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 613-237-5160
(B) TELEFAX: 613-787-3558
I
CA 02257867 1999-10-04
54
(2) INFORMATION FOR SEQUENCE ID N0:1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 1
cattcatgac tttgggatca atatcagcat atgcagttca tccaacgata aacttaatcc 60
gaattataga gc 72
(2) INFORMATION FOR SEQUENCE ID N0:2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 2
cggtctgaat tcaattcaac attttgttcg ttcgggtttg attgtgtcgt agctctataa 60
ttcggattaa g 71
(2) INFORMATION FOR SEQUENCE ID NO: 3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:24
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 3
caatatcagc aatgcagttc atcc 24
CA 02257867 1999-10-04
(2) INFORMATION FOR SEQUENCE ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:24
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 4
ccaagcgatc ttcttcttgt ccaa 24
(2) INFORMATION FOR SEQUENCE ID NO: 5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:18
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 5
gcgctcgatg acgccaac 1g
(2) INFORMATION FOR SEQUENCE ID N0: 6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 6
cacgacgaga tcctcgccg 1g
CA 02257867 1999-10-04
56
(2) INFORMATION FOR SEQUENCE IS NO: 7
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 7
gaatagcctc tccaccca 1g
(2) INFORMATION FOR SEQUENCE ID NO: 8
(i) SEQUENCE CHARACTERICTICS:
(A) LENGTH:24
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 8
ggaatccttc cagtagtatc ggcc 24
(2) INFORMATION FOR SEQUENCE ID NO: 9
(i) SEQUENCE CHARACTERICTICS:
(A) LENGTH:21
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 9
ggaaaataaa acagcaagta c 21
CA 02257867 1999-10-04
57
(2) INFORMATION FOR SEQUENCE ID N0: 10
(i) SEQUENCE CHARACTERICTICS:
(A) LENGTH:20
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 10
caaatattgc aaagtcccgg 20
(2) INFORMATION FOR SEQUENCE ID NO: 11
(i) SEQUENCE CHARACTERICTICS:
(A) LENGTH:18
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 11
ccggatcgcc acctacac 18
(2) INFORMATION FOR SEQUENCE ID N0: 12
(i) SEQUENCE CHARACTERICTICS:
(A) LENGTH:20
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 12
tggctataac agagtttctc 20
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58
(2) INFORMATION FOR SEQUENCE ID NO: 13
(i) SEQUENCE CHARACTERICTICS:
(A) LENGTH:21
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 13
ggatttccag aagaagatgc c 21
(2) INFORMATION FOR SEQUENCE ID NO: 14
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:21
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 14
gttcgttcgg gtttgattgt g 21
(2) INFORMATION FOR SEQUENCE ID NO: 15
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:21
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 15
gaactcaacg ggcccttccc c 21
CA 02257867 1999-10-04
59
(2) INFORMATION FOR SEQUENCE ID NO: 16
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:24
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 16
ggagggaagt ggagtaaatg gccg 24
(2) INFORMATION FOR SEQUENCE ID N0: 17
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:24
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 17
caatatcagc aatgcagttc atcc 24
(2) INFORMATION FOR SEQUENCE ID NO: 18
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:19
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 18
cacgacgaga tcctcgccg 19
CA 02257867 1999-10-04
(2) INFORMATION FOR SEQUENCE ID NO: 19
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:18
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 19
gaatagcctc tccaccca 1g
(2) INFORMATION FOR SEQUENCE ID NO: 20
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:24
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 20
ggaatccttc cagtagtatc ggcc 24
(2) INFORMATION FOR SEQUENCE ID NO: 21
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:21
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 21
ggaaaataaa acagcaagta c 21
CA 02257867 1999-10-04
61
(2) INFORMATION FOR SEQUENCE ID NO: 22
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:31
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 22
aattcgaagc gcttggatac agttgtaggg a 31
(2) INFORMATION FOR SEQUENCE ID N0: 23
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:23
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 23
gtaagagatg tgaatccgcc tgt 23
(2) INFORMATION FOR SEQUENCE ID N0: 24
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:20
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 24
gcataagtcg ttagaaggag 20
' ~ CA 02257867 1999-10-04
62
(2) INFORMATION FOR SEQUENCE ID NO: 25
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:22
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 25
gaagaaagaa aattaaggaa cc 22
(2) INFORMATION FOR SEQUENCE ID NO: 26
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:37
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 26
catgccatgg ctagcattga acaagatgga ttgcacg 37
(2) INFORMATION FOR SEQUENCE ID NO: 27
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:28
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 27
gtactctaga cccgctcaga agaactcg 28
CA 02257867 1999-10-04
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(2) INFORMATION FOR SEQUENCE ID NO: 28
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:33
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 28
ctagccatgg ctttgggatc aatatcagca atg 33
(2) INFORMATION FOR SEQUENCE ID NO: 29
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:31
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 29
ctagccatgg catcagcaat gcagttcatc c 31
(2) INFORMATION FOR SEQUENCE ID NO: 30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:33
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 30
ctagccatgg cgttcatcca acgataaact taa 33
CA 02257867 1999-10-04
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(2) INFORMATION FOR SEQUENCE ID N0: 31
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:35
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 31
ctagccatgg cataaactta atccgaatta tagag 35
(2) INFORMATION FOR SEQUENCE ID NO: 32
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:32
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 32
ctagccatgg ccgaattata gagctacgac ac 32
(2) INFORMATION FOR SEQUENCE ID NO: 33
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:31
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 33
ctagccatgg ctacgacaca atcaaacccg a 31
CA 02257867 1999-10-04
(2) INFORMATION FOR SEQUENCE ID NO: 34
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:30
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 34
ctagctagct tcaacatttt gttcgttcgg 30
(2) INFORMATION FOR SEQUENCE ID N0: 35
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:29
(B) TYPE:: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO 35
ctagctagct tcgttcgggt ttgattgtg 29
(2) INFORMATION FOR SEQUENCE ID NO: 36
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 36
ctagctagct gattgtgtcg tagctctata 30
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(2) INFORMATION FOR SEQUENCE ID NO: 37
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 37
ctagctagcc gtagctctat aattcggatt 30
(2) INFORMATION FOR SEQUENCE ID NO: 38
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 38
ctagccatgg tatagagcta cgacac 26
(2) INFORMATION FOR SEQUENCE ID NO: 39
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 39
ctagctagca agtgtcgtag ctctata 27
' CA 02257867 1999-10-04
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(2) INFORMATION FOR SEQUENCE ID NO: 40
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 40
aattatagag ctccgacaca atc 23
(2) INFORMATION FOR SEQUENCE ID NO: 41
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 41
aattatagag ctaccacaca atc 23
(2) INFORMATION FOR SEQUENCE ID N0: 42
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 42
ctagccatgg tattttgagc acgggttttt ctggtcc 37
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(2) INFORMATION FOR SEQUENCE ID NO: 43
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID N0: 43
ctagctagct ggaaaaacta caattattgt taacc 35
(2) INFORMATION FOR SEQUENCE ID NO: 44
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID N0: 44
ctagccatgg aaacgaacta aaggagaata c 31
(2) INFORMATION FOR SEQUENCE ID NO: 45
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 45
ctagctagcc gggataggtg ttttgtataa a 31
CA 02257867 1999-10-04
69
(2) INFORMATION FOR SEQUENCE ID NO: 46
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 46
ctagccatgg cagctactct agggggaatg 30
(2) INFORMATION FOR SEQUENCE ID N0: 47
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 47
ctagtctaga cgtatacgtc aggagtcca 29
(2) INFORMATION FOR SEQUENCE ID NO: 48
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID N0: 48
atggtcccgg t 11
CA 02257867 1999-10-04
(2) INFORMATION FOR SEQUENCE ID NO: 49
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 49
ctagccatgg gtcccggtat ttattaccg 29
(2) INFORMATION FOR SEQUENCE ID NO: 50
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: Sequence source:/note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 50
ctaggtcgac ttaggcattt tcttttgacc caat 34
(2) INFORMATION FOR SEQUENCE ID NO: 51
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 259
(B) TYPE: DNA
(ii) ORGANISM: Nicotiana tabacum
SEQUENCE DESCRIPTION: SEQ ID NO: 51
atgactatagatcgaacctatccaatttttacagtacgatggttggctgttcacggccta60
gctgtacctaccgtcttttttttgggatcaatatcagcaatgcagttcatccaacgataa120
acttaatccgaattatagagctacgacacaatcaaacccgaacgaacaaaatgttgaatt180
gaatcgtaccagtctctactgggggttattactcatttttgtacttgctgttttattttc240
caattatttcttcaattaa 259
CA 02257867 1999-10-04
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(2) INFORMATION FOR SEQUENCE ID N0: 52
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40
(B) TYPE: PRT
(ii) ORGANISM: Nicotiana tabacum
SEQUENCE DESCRIPTION: SEQ ID N0: 52
Met Thr Ile Asp Arg Thr Tyr Pro Ile Phe Thr Val Arg Trp Leu Ala
1 5 10 15
Val His Gly Leu Ala Val Pro Thr Val Phe Phe Ile Leu Gly Ser Ile
20 25 30
Ser Ala Met Gln Phe Ile Gln Arg
35 40
(2) INFORMATION FOR SEQUENCE ID NO: 53
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38
(B) TYPE: PRT
(ii) ORGANISM: Nicotiana tabacum
SEQUENCE DESCRIPTION: SEQ ID NO: 53
Met Thr Gln Ser Asn Pro Asn Glu Gln Asn Val Glu Leu Asn Arg Thr
1 5 10 15
Ser Leu Tyr Trp Gly Leu Leu Leu Ile Phe Val Leu Ala Val Leu Phe
20 25 30
Ser Asn Tyr Phe Phe Asn
(2) INFORMATION FOR SEQUENCE ID N0: 54
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300
(B) TYPE: DNA
ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 54
gagctcggta cccaaagctc ccccgccgtc gttcaatgag aatggataag aggctcgtgg 60
gattgacgtg agggggcagg gatggctata tttctgggag cgaactccgg gcgaatacga 120
agcgcttgga tacagttgta gggagggatc catgactttg ggatcaatat cagcaatgca 180
gttcatccaa cgataaactt aatccgaatt atagagctac gacacaatca aacccgaacg 240
aacaaaatgt tgaaggggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag 300
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72
(2) INFORMATION FOR SEQUENCE ID NO: 55
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 55
Met Thr Leu Gly Ser Ile Ser Ala Met Gln Phe Ile Gln Arg
1 5 10
(2) INFORMATION FOR SEQUENCE ID NO: 56
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 56
Met Thr Leu Gln Ser Asn Pro Asn Glu Gln Asn Val Glu Gly Glu Ala
1 5 10 15
Val Ile Ala Glu Val Ser Thr Gln Leu Ser Glu Val
20 25
(2) INFORMATION FOR SEQUENCE ID NO: 57
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="syntheic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 57
gagctcggta cccaaagctc ccccgccgtc gttcaatgag aatggataag aggctcgtgg 60
gattgacgtg agggggcagg gatggctata tttctgggag cgaactccgg gcgaatacga 120
agcgcttgga tacagttgta gggagggatc catgactttg ggatcaatat cagcaatgca 180
gttcatccaa cgataaactt aatccgaatt atagagctac gacacaatca aacccgaacg 240
aacaaaatgt tgaattgggg attgaacaag atggattgca cgcaggttct ccggccgctt 300
CA 02257867 1999-10-04
73
(2) INFORMATION FOR SEQUENCE ID N0: 58
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 58
Met Thr Leu Gly Ser Ile Ser Ala Met Gln Phe Ile Gln Arg
1 5 10
(2) INFORMATION FOR SEQUENCE ID NO: 59
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 59
Met Thr Gln Ser Asn Pro Asn Glu
1 5
(2) INFORMATION FOR SEQUENCE ID NO: 60
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 60
Met Leu Asn Trp Gly Leu Asn Lys Met Asp Cys Thr Gln Val Leu Arg
1 5 10 15
Pro Leu Gln Asn Val Glu Leu Gly Ile Glu Gln Asp Gly Leu His Ala
20 25 30
Gly Ser Pro Ala Ala Trp
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74
(2) INFORMATION FOR SEQUENCE ID N0: 61
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 61
ccgaattata gagctacgac ac 22
(2) INFORMATION FOR SEQUENCE ID NO: 62
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 62
aatattttga gcacgggttt ttatggtcca agtgtatctt gtctttacta cgaattattt 60
tccttggtta acaataattg tagtttttcc aatatttgcg ggttccttaa ttttctttct 120
tccccataaa ggaaataggg taattaggtg gtatacgata tgtatatgta ttttagaact 180
(2) INFORMATION FOR SEQUENCE ID NO: 63
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 63
Met Asn Tyr Phe Pro Trp Leu Thr Ile Ile Val Val Phe Pro Ile Phe
1 5 10 15
Ala Gly Ser Leu Ile Phe Phe Leu Pro His Lys Gly Asn Arg Val Ile
20 25 30
Arg Trp Tyr Thr Ile Cys Ile Cys Ile Leu Glu
35 40
CA 02257867 1999-10-04
7S
(2) INFORMATION FOR SEQUENCE ID NO: 64
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 171
(B) TYPE: DNA
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 64
tccacttcta gatagagaaa cgaactaaag gagaatactt aataatacgg cgaaacattt 60
atacaaaaca cctatcccga gcacacgcaa gggaaccgta gacaggcaag tgaaatccaa 120
tccacgaaat aaattgatcc atggacggca ccgttgtggt aaaggtcgta a 171
(2) INFORMATION FOR SEQUENCE ID N0: 65
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 65
Pro Leu Leu Asp Arg Glu Thr Asn
1 5
(2) INFORMATION FOR SEQUENCE ID NO: 66
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41
(B) TYPE: PRT
(ii) ORGANISM: Artificial Sequence
ADDITIONAL INFORMATION: /note="synthetic sequence"
SEQUENCE DESCRIPTION: SEQ ID NO: 66
Met Ala Lys His Leu Tyr Lys Thr Pro Ile Pro Ser Thr Arg Lys Gly
1 5 10 15
Thr Val Asp Arg Gln Val Lys Ser Asn Pro Arg Asn Lys Leu Ile His
20 25 30
Gly Arg His Arg Cys Gly Lys Gly Arg
35 40
