Note: Descriptions are shown in the official language in which they were submitted.
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PLANT TRANSCRIPTION REGULATORS FROM CIRCOVIRUS
The present invention relates generally to a novel range of transcription
regulators and
transcription regulator-like sequences operable in plants. More particularly,
the present
invention is directed to transcription regulators and transcription regulator-
like sequences
of viral origin and, even more particularly, of circovirus origin. The
transcription
regulators and transcription regulator-like sequences of the instant invention
are useful
in genetic engineering of plants and in particular leguminous plants such as
to facilitate
or control expression of foreign genes. The transcription regulators and
transcription
regulator-like sequences of the present invention are also useful in
facilitating different
levels of expression in different plant tissue types.
Bibliographic details of the publications referred by author in this
specification are
collected at the end of the description. Sequence Identity Numbers (SEQ ID
Nos.) for
the nucleotide sequences referred to in the specification are defined
following the
bibliography.
Throughout this specification, unless the context requires otherwise, the word
"comprise", or variations such as "comprises" or "comprising", will be
understood to
imply the inclusion of a stated element or integer or group of elements or
integers but
not the exclusion of any other element or integer or group of elements or
integers.
Transcription regulators are molecules, and generally nucleotide-based
molecules, which
facilitate and modulate expression of genetic sequences at the level of
transcription.
Transcription regulators include promoters and termination and polyadenylation
sequences amongst other effectors and facilitators of transcription.
Promoters are specific nucleotide sequences to which RNA polymerase binds to
initiate
RNA synthesis in cells. They contain the start site for RNA synthesis and the
genetic
signals to initiate polymerase mediated RNA synthesis. In addition, sequence
specific
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DNA-binding proteins are presumed either to inhibit or to
stimulate the initiation of RNA synthesis by binding next to
the promoter and affecting the binding of the polymerase to
the promoter. Terminator sequences refer to termination and
polyadenylation sequences and are required for transcription
of functional mRNAs. Termination sequences are located
downstream (i.e. at the 3' end) of a gene and are recognized
by RNA polymerase as a signal to stop synthesizing mRNA.
Polyadenylation sequences are signals required for
polyadenylation of eukaryotic mRNA molecules following
transcription.
A viral promoter widely used to facilitate foreign
gene expression in plants is the cauliflower mosaic virus
(CaMV) 35S promoter (Odell et al. 1985), which is derived
from a double-stranded DNA plant virus. The use of this
promoter in plants and plant cells is well documented
(Benfey and Chua, 1990; Higgins and Spencer, 1991).
However, despite the apparent usefulness of this promoter,
it is not functional in all plants and is particularly
poorly operable in leguminous plants. There is a need,
therefore, to identify other promoters, such as of viral
origin, which are operable in plants and particularly
leguminous plants. There is also a need to modulate levels
of expression of genes and other genetic sequences within
plant cells.
Accordingly, one aspect of the present invention
is directed to a genetic construct comprising a circovirus
promoter or promoter-like sequence and which is operable in
a plant cell wherein said circovirus comprises a
multicomponent DNA genome.
In a related aspect of the present invention,
there is provided a genetic construct comprising a
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circovirus promoter or promoter-like sequence and a
termination and/or polyadenylation sequence, which sequences
are operable in a plant cell.
In one aspect, there is described a genetic
construct comprising: a circovirus promoter or promoter-like
sequence from a circovirus which comprises a multicomponent
DNA genome, said promoter or promoter-like sequence being
operable in a plant cell, wherein said promoter is obtained
from one of segments 1 to 7 of subterranean clover stunt
virus (SCSV) (SEQ ID NOs: 1 to 7), and wherein said
promoter-like sequence has a substantially identical
sequence to the promoter obtained from one of segments
1 to 7 of SCSV and has promoter activity in a plant cell;
and a heterologous gene operably linked to said promoter or
promoter-like sequence.
In another aspect, there is described a genetic
construct comprising an SCSV promoter or promoter-like
sequence operable in a plant cell, and a heterologous gene
inserted at a restriction endonuclease site downstream of
said promoter or promoter-like sequence and operably linked
to said promoter or promoter-like sequence, wherein said
promoter is obtained from one of segments 1 to 7 of SCSV
(SEQ ID NOs: 1 to 7), and wherein said promoter-like
sequence has a substantially identical sequence to the
promoter obtained from one of segments 1 to 7 of SCSV and
has promoter activity in a plant cell.
In another aspect, there is described a genetic
construct comprising at least two heterologous genes
operably linked to the same or different circovirus
promoters or promoter-like sequences, each promoter or
promoter-like sequence being from a circovirus which
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comprises a multicomponent DNA genome and being operable in
a plant cell, wherein each said promoter is obtained from
one of segments 1 to 7 of SCSV (SEQ ID NOs: 1 to 7), and
wherein said promoter-like sequence has a substantially
identical sequence to the promoter obtained from one of
segments 1 to 7 of SCSV and has promoter activity in a plant
cell.
In another aspect, there is described a method of
expressing a foreign gene in a plant cell, said method
comprising transforming said plant cell with the genetic
construct of the invention.
A circovirus is a non-geminated single stranded
(ss) DNA plant virus (Table 1), distinct from caulimoviruses
which have a double stranded genome, and geminiviruses, the
only other known ssDNA plant viruses, which have geminated
particles (Chu et al., 1994). As used herein, the
circovirus group is considered to include subterranean
clover stunt virus (SCSV) (Chu and Helms, 1988), banana
bunchy top virus (BBTV) (Thomas and Dietzgen,
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1991; Handing et al. 1991; 1993; Burns et al. 1993) and milk-vetch dwarf virus
(MDV)
(Isogai et al. 1992; Sano et al. 1993) and faba bean necrotic yellows virus
(FBNYV)
(Katul et al., 1993).
In a particularly preferred embodiment, the circovirus contemplated for use in
accordance
with the present invention comprises more than two DNA components or segments.
The present invention is particularly directed and hereinafter described with
reference to
~b~ clover stunt virus (hereinafter abbreviated to "SCSV') as a representative
of
IO the circovirus group. This is done, however, with the understanding that
reference to
SCSV includes reference to all other suitable members of the circovirus group
to which the
instant invention extends. Preferred members of the circovirus group such as
SCSV
comprise more than two DNA components or segments. Reference hereinafter to
"SCSV"
also includes and extends to all naturally occurring or artificially induced
mutants,
derivatives, parts, fragments, homologues or analogues of the virus which
still retain at
least one suitable promoter and/or termination and/or polyadenylation
sequences.
Accordingly, a particularly preferred embodiment of the present invention
contemplates
a genetic construct comprising an SCSV promoter or promoter-like sequence and
which
is operable in a plant cell.
A related aspect of the present invention is directed to a genetic construct
comprising an
SCSV promoter or promoter-like sequence and a termination and/or
polyadenylation
sequence, which sequences are operable in a plant cell.
The term "genetic construct" is used in its broadest sense to include any
recombinant
nucleic acid molecule such as an isolated nucleic acid molecule, vector,
expression vector
or binary vector. It may comprise solely the circovirus promoter or may
contain one or
more promoters in association with regulatory and/or reporter sequences.
AMENL~E~ SHEET
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The genetic construct may be double or single stranded DNA, in linear or
covalently
closed circular form. As stated above, it may comprise only the promoter or
promoter-
like sequence or may carry other heterologous or homologous transcription
regulator
sequences and/or heterologous structural gene sequences including promoters
associated
with SCSV. By "homologous" is meant a gene sequence naturally associated with
the
SCSV.promoter. "Heterologous" means a "foreign" gene relative to the promoter
or a
gene not otherwise normally associated with the SCSV promoter. In a preferred
embodiment, the foreign gene is also foreign to SCSV. Examples of foreign
genes
include genes which facilitate resistance to insects or other pest
infestation, enhance
resistance to insecticides or herbicides, promote frost resistance, alter
flower or petal
colour, decrease the rate of senescence, especially in cut flowers, increase
or enhance
levels of certain proteins and/or ribozymes. More particularly, the foreign
genes include:
a) a resistance gene against plant viruses, bacteria, fungi, nematode and
other pathogens;
b) a plant virus resistance gene including a synthetic gene from and against
alfalfa mosaic virus, subterranean clover stunt virus, subterranean clover
mottle virus, subterranean clover red leaf virus, potato leafroll virus,
tomato spotted wilt virus, bean yellow mosaic virus, white clover mosaic
virus, clover yellow vein virus, potato viruses x, y, s, m and a, cucumber
mosaic virus, rice ragged stunt virus and barley yellow dwarf virus;
c) a gene to improve nutritional value of plants such as sunflower high
sulphur gene SFB;
d) a bloat resistance gene;
e) an antibody gene;
f) a cereal thionin and ribosome inactivating protein gene;
g) an insect resistance gene including BT toxin gene and proteinase inhibitor
gene from Nicotiana alata;
h) a selectable marker gene such as those conferring resistance to
kanamycin, phosphinothricin, spectinomycin and hygromycin;
i) a reporter gene such as GUS, CAT and pigment genes;
j) a gene encoding a regulatory protein which modulates expression of a
gene in plant cells.
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The present invention further contemplates a genetic construct comprising two
heterologous genes operably linked to the same or different circovirus
promoters
operable in a plant cell. Preferaby the promoter or different promoters are
from a
' circovirus with a genome comprising more than two components or segments.
Most
preferably the promoter or different promoters are from SCSV and in particular
are
selected from segments 1 to 7 of SCSV as defined by SEQ ID NOs. 1 to 7. (See
below).
The genetic constructs may also comprise a termination or polyadenylation
sequence
operably linked to one or both of the heterologous genes. In one embodiment,
the
termination and/or polyadenylation sequence is the same for each gene. In an
alternative
embodiment the termination and/or polyadenylation sequence is different for
each gene.
Most preferably the termination and polyadenylation sequence is selected from
segments
1 to 7 of SCSV as defined by SEQ ID NOs. 1 to 7 (See below). In yet another
embodiment at least one termination and polyadenylation sequence is from the
MeA 3
gene of Flaveria bidentis (see below).
The term "transcription regulator" is used in its broadest sense to include
promoters,
termination and polyadenylation sequences and other effectors and facilitators
of
transcription of genetic sequences. As with promoters contemplated by the
present
inveniton, the termination and polyadenylation sequences may be of SCSV origin
and
may be naturally associated with a corresponding promoter from SCSV or may be
associated with another promoter of SCSV. Alternatively, the termination
and/or
polyadenylation sequences may be derived from non-SCSV sources. A particularly
preferred terminator comprises the 3' nucleotide sequence of the MeA3 gene of
Flaveria
bidentis which codes for an NADP-malic enzyme of C4 photosynthesis (Hatchi,
1987).
The nucleotide sequence of the terminator region of the MeA gene is shown in
Figure
15. The combination of an SCSV promoter with, for example, the F. bidentis MeA
gene terminator sequence results in a high level of expression especially in
monocotyledonas plants relative to constructs without the terminator sequence.
r
The foreign gene may also be in the antisense orientation so as to facilitate
reduced
levels of endogenous plant gene products. In this regard, "gene" may be ten
base pairs
in length, tens of base pairs in length, hundreds of base pairs in length or a
full length
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or near full length gene but in a reverse orientation relative the promoter.
The foreign
gene may also be placed in the "sense" orientation for co-suppression of a
target gene.
According to another aspect of the present invention, there is provided a
genetic
construct comprising an SCSV promoter or promoter-like sequence operable in a
plant
cell and at least one restriction endonuclease site downstream of said
promoter to
facilitate insertion of a heterologous gene such that said gene is operably
linked to said
promoter. In an alternative embodiment, the genetic construct comprises an
SCSV
promoter or promoter-like sequence operable in a plant cell and a heterologous
gene
operably linked to said promoter. In both embodiments, the term "gene"
includes those
directing the synthesis of oligonucleotides such as those useful in antisense
techniques
as well as ribozymes.
In still a further embodiment of the present invention, there is contemplated
a genetic
construct comprising an SCSV promoter or promoter-like sequence operable in a
plant
cell and at least one restriction endonuclease site downstream of said
promoter to
facilitate insertion of a heterologous gene such that said gene is operably
linked to said
promoter and a termination and/or polyadenylation sequence positioned such
that same
sequence is at the 3' end relative to said heterologous gene to facilitate
expression of
said heterologous gene. Preferably, the termination sequence is from SCSV.
Alternatively, the terminator sequence is from the F. bidentis MeA3 gene.
Plants contemplated by the present invention include both monocotyledonous and
dicotyledonous species. The present invention also extends to leguminous and
non-
leguminous plants although leguminous plants are preferred.
The SCSV genome comprises at least seven distinct circular ssDNA components
described as segments 1-7. The size of these segments range from about 988 to
about
1022 nucleotides. Each of the seven DNA components of SCSV contains one major
open reading frame of the viral sense and a non-coding region of various
lengths
containing a conserved potential stem and loop structure (Figures l and 2;
Table 2).
Each transcription unit contains a typical TATA box and a polyadenylation
signal for
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start and end of transcription, respectively (Figure 2, Table 2).
Because the DNAs are circular, the sequences in the non-coding regions
comprise the
promoters and the terminator signals which vary with different DNA components
(Table
2). The TATA boxes and the stem-loops of the two replicase-associated protein
genes
in segments 2 and 6 are quite different from those of the other genes. In
contrast, the
stem-loops and TATA boxes are the same in segments 1, 3, 4, 5 and 7. All the
DNAs,
except those of segments 2 and 6, also share a common sequence (known as the
common region) in the non-coding region (Figures 1 and 2).
The present invention, therefore, extends to each of the seven promoters and
to
termination and polyadenylation sequences on segments 1-7 of SCSV. The
nucleotide
sequences of segments 1-7 are shown in Figure 6 and are defined in SEQ ID NOs.
1-7,
respectively.
Segment S was identified as the coat protein gene based on N-terminal amino
acid
sequence and amino acid composition data (Chu et al., 1993a). Segments 2 and 6
encode proteins containing the characteristic NTP-binding motifs and thus are
predicted
to be the putative viral replication-associated protein (RAP) genes. The
remaining 4
DNA components are unrelated to each other or to segment 2, 5 and 6, based on
their
distinctive deduced amino acid sequence. The SCSV DNAs have no significant
nucleotide sequence homology with the genomes of geminiviruses although some
homology exists at the deduced amino acid level.
The replicative competency of SCSV has been demonstrated (Chu et al., 1993b).
Since
the SCSV virion DNA is single-stranded and the transcripts are of viral sense,
the first
likely biosynthetic event after uncoating is likely to be the synthesis of the
replicative
form DNA using host DNA polymerase. (The DNAs of SCSV have the ability to self
prime in dsDNA synthesis [Chu and Helms, 1988)). Host RNA polymerase is
thought
to bind to the promoters initiating RNA transcription followed by synthesis of
the viral
a
proteins required for virus multiplication.
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In a particularly preferred embodiment of the present invention, there is
contemplated
an SCSV promoter or promoter-like sequence comprising a nucleotide sequence
selected
from within SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID N0: 4, SEQ ID
NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 and/or genetic constructs comprising
same.
A "promoter-like sequence" as used herein includes any functional mutant,
derivative,
part, fragment, homologue or analogue of a naturally occurring SCSV promoter.
Promoter-like sequences contemplated herein include single or multiple
nucleotide
substitutions, deletions and/or additions to an SCSV promoter, provided that
the said
promoter-like sequences retain at least 35%, prererably at least 45%, more
preferably
at least 55%, even more preferably at least 65-70% and still more preferably
at least 85-
95% or greater promoter activity compared with the corresponding wild-type
SCSV
promoter.
In yet another embodiment, there is provided a genetic construct comprising an
SCSV
promoter or promoter-like sequence and which is operable in a plant cell, said
promoter
or promoter-like sequence corresponding to all or part of any one of SEQ ID
NO: 1 to
7 or capable of hybridising under a range of stringency conditions, ranging
from high
to low stringency conditions to at least one of SEQ ID NO: 1 to 7.
Conveniently, a
mutant, derivative, part, fragment, homologue or analogue of an SCSV promoter
is
defined as being functional in a plant cell and capable of hybridising under a
range of
at least high to low stringency conditions to at least one of SEQ ID NO: l,to
7.
In a particularly preferred embodiment, the genetic construct further
comprises a
termination and/or polyadenylation sequence from SCSV or from non-SCSV source
and
which enhances or other facilitates expression of a gene operably linked to
said
promoter.
For the purposes of defusing the level of stringency, reference can
conveniently be made
to Sambrook et al (1989) where the washing steps at pages 9.52-9.57 are
considered high stringency. A low stringency is defined herein as being in
0.1-0.5% w/v SDS at 37-45°C for 2-3 hours. Depending on the
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source and concentration of nucleic acid involved in the hybridisation,
alternative
conditions of stringency may be employed such as medium stringent conditions
which
are considered herein to be 0.25-0.5% w/v SDS at >_ 45°C for 2-3 hours
or high
stringent conditions as disclosed by Sambrook et al (1989).
Another embodiment of the present invention contemplates a method of
expressing a
foreign gene in a plant cell, said method comprising introducing into said
plant cell a
genetic construct comprising and SCSV promoter or promoter-like sequence
operable
in said plant cell and operably linked to said foreign gene. In a further
embodiment,
multiple SCSV promoters are used to drive one or more transgenes without
antagonism.
In yet a further embodiment, the SCSV promoter is associated with the SCSV
segment
2 gene in order to enhance the expression of the foreign gene. In another
embodiment,
the genetic constructs further comprises one or more termination and/or
polyadenylation
sequences which are located at the 3' end of the foreign gene. These sequences
enhance
or otherwise facilitate expression of the foreign gene. The termination and/or
polyadenylation segment may be from SCSV or from another source such as the F.
bidentis MeA gene.
In still yet another embodiment, the present invention contemplates a
transgenic plant
carrying an SCSV promoter or promoter-like sequence as hereinbefore defined in
its
genome and optionally a termination and/or polyadenylation sequence to enhance
expression of a gene downstream of said promoter. Preferably, the transgenic
plant
exhibits altered characteristics due to expression of a genetic sequence such
as a gene,
oligonucleotide or ribozyme downstream of the SCSV promoter.
The present invention is further described by reference to the following non-
limiting
figures and/or examples. Reference herein to a promoter region from SCSV is
abbreviated to "S" for SCSV, the genome segment number (e.g. 1, 3, 4, 5 and 7)
and
"nc" for non-coding region. For example, the promoter from SCSV genome segment
1 is defined as "Slnc". Terminator sequences for particular SCSV genome
segments are
indicated for example, as follows: "SCITr" or "SCSTr" for the terminator
sequences for
segments 1 and 5, respectively. Genetic constructs comprising an SCSV
promoter, a
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reporter gene such as GUS and a terminator sequence such as from SCSV is
abbreviated
to "SCSV:GUS:SCTr" or "SCSV:GUS:SCSVTr". Specific promoters and termination
sequences are defined as above, for example S4nc:GL'S:SCITr or S4nc:GUS:Me3",
"S4nc:GUS:Me3". In the latter construct the terminator sequence from the MeA
gene
of Flaveria bidentis is used, referred to herein as "Me3".
In the Figures:
FIGURE 1 shows the structures and transcription units found in a
representative DNA
component of a typical geminivirus and SCSV, both of which contain a ssDNA
genome.
FIGURE 2 shows the seven DNA segments found in the genome of SCSV in a linear
form, indicating the positions of the stem-loop structure, the common region,
the open
reading frame (ORF), the TATA box and the termination and polyadenylation
signals
on each DNA.
FIGURE 3 shows the construction of the seven SCSV DNA non-coding region: ~i-
glucuronidase (GUS) fusion expression vectors for transformation into tobacco
plants.
The amplified PCR fragments were separately cloned in front of the GUS gene in
pHW9
at the BamHI (B) and NcoI (I~ sites as indicated. The resultant recombinant
pHW9
vectors were cut at the EcoRI site and cloned into the EcoRI site of the
recipient
PGA470 binary vector.
FIGURE 4 shows the construction of the segments 5 and 7 promoter:GUS fusion
expression vectors and their deletion derivatives for protoplast studies. DNAs
corresponding to the full-length non-coding regions were obtained by PCR and
cloned
into pKGO in front of GUS by blunt end ligation at the SaII site as indicated.
The
deletion derivatives were obtained by digesting the pKGO clones containing the
full-length sequence with HindIII or PstI on the vector and the appropriate
restriction
enzymes on the SCSV sequence as indicated. The deleted pKGO constructs were
religated after end-filling as required.
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FIGURE 5 shows the construction of the recombinant binary pTAB 10 vector (pBS
150)
containing the seg 7 promoter(57nc):GUS fusion gene for transformation into
subterranean clover plants. The 57nc:GUS expression cassette was excised from
the
pKGO construct (Fig. 4) by digestion with HindIII and BamHI and blunt-end
ligated to
pTAB 10 at the EcoRI site after end-filling the DNAs.
FIGURE 6 shows the complete sequences of the seven SCSV DNA circles. The
sequence of the non-coding region on each DNA used in the construction of the
expression cassettes are underlined.
FIGURE 7 shows GUS expression detected by histochemical staining on leaves of
transgenic tobacco and subterranean clover transformed with the SCSV seg 7
promoter(S7nc):GUS fusion expression cassette.
FIGURES 8A to 8F are photographic representations of histochemical staining
for GUS
activity in transgenic plants - bright field. Bright field exposures of
stained leaf pieces
(L), stem sections (S), roots (R) and pollen (P) from tobacco plants
transformed with the
GUS fusion constructs containing the SCSV promoter regions from segments 1, 3,
4, 5
and 7 referred to as Slnc, S3nc, S4nc, SSnc and S7nc promoter regions and from
non-
transformed plants (NT). Blue colouration indicates GUS expression. Each leaf,
stem
or root piece represents an independent transformant (except the top two root
pieces of
SSnc which could not be separated easily) and the pollen samples were mixtures
from
two or more transformants. Fig. 8a shows differential expression of GUS in
plants
transformed with either S4nc or SSnc regions compared to a non-transformed
plant
(I~.
FIGURES 9A to 9E are photographic representations showing histochemical
staining
for GUS activity in transgenic plants - dark field. Transverse (T) and
longitudinal(L)
4
thin sections of stained, embedded stem pieces from tobacco plants transformed
with the
GUS fusion constructs containing the component 1 (Slnc), 3 (S3nc), 4 (S4nc), 5
(SSnc),
and 7 (S7nc) promoter regions, viewed with a dark field. Pink crystals
indicate GUS
expression. The stem pieces used for the transverse and longitudinal sections
represent
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independent transformants. The magnifications are: 375X in Slnc T and L, S3nc
L,
S4nc T, SSnc T and L, and S7nc L; 480X in S3nc T; 200X in S4nc L; and 300X in
S7nc T.
FIGURE 10 is a graphical representation of a fluorometric assay for GUS
expression.
Fluorometric assay results from leaf extracts of tobacco plants transformed
with the GUS
fusion constructs containing the component 1 (Slnc), 3 (S3nc), 4, (S4nc), 5
(SSnc), and
7 (S7nc) promoter regions. Each column represents an independent transformant.
GUS
activities were measured with a Labsystems Fluoroskan at 5 or 10 minute
intervals over
60 minutes (or 30 minutes for S4nc) using 4-methylumbelliferyl ~i-D-
glucuronide
(MUG) as the substrate. The rates of GUS activity are expressed as
fluorometric units
(F1.U.) per minute per mg of protein. 1000 F1.U is approximately equal to 825
pmoles
of 4-methylumbelliferone (MLn.
FIGURE 11 is a diagramatic representation of the SCSV segment 2 promoter
(S2nc)
construct capable of directing GUS expression in tobacco protoplasts. A
fragment of
segment 2 DNA from NcoI XbaI was fused to the promoterless GUS vector, pKGO.
Pr,
promoter, seg 2, segment 2 of SCSV.
FIGURES 12a and 12b are diagramatic representations showing constructs of
SCSV:GUS:SCSV Tr expression vectors. The termination/polyadenylation sequences
for segment 3 of SCSV (SC3Tr) and segment 5 (SCSTr) were amplified by PCR and
cut with the respective restriction enzymes and then cloned into recombinant
pKGO
vector as indicated. The SC3Tr construct was cloned as an EcoRI XhoI fragment
into
the pKGo vector containing Slnc:GUS:OCS3' to make Slnc:GUS:SC3Tr. The SSCSTR
construct was made as an EcoRV fragment into Vector pKGO containing
S4nc:Gus:OCS3' to make S4nc:GUS:SSTr.
FIGURES 13A to 13D are photographic representations showing GUS expression in
potato plant tissues directed by the SCSV segment 4 promoter (S4nc). A. stem;
B, leaf;
C. stolon; and D. tuber.
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FIGURE 14 is a photographic representation of GUS expression in cotton leaf
directed
by the SCSV segment 7 promoter (S7nc).
- FIGURE 15 is a representation of the MeA3's terminator sequence of the
Flaveria
bidentis MeA gene (Me3). The stop codon is shown in bold face type at the
beginning
of the sequence. This sequence wads engineered in the Chimeric construct to
include
an EcoRl site: GAATTCGTTTAG.... The chimeric constructs thus contained a
sequence begining AATTCGTTTAG.
FIGURE 16 is a diagramatic representation of GUS expression vectors (ME20 and
ME29) containing the indicated Flaveria bidentis MeA gene regulatory elements.
FIGURE 17 is a diagramatic representation of the construction of S4nc plasmid
pBS237
containing the expression cassette S4nc:GUS:Me3'. Plasmid pBS218 was digested
with
EcoRl to remove OCS3' region and ligated with an EcoRl.
FIGURE 18 is a diagramatic representation of the construction of plasmid
pBS246
containing the Slnc:nptII:SC3Tr expression cassette. The SaII-SaII fragment is
approximately 8.5-9 khp; BL is about 0.5 kbp; BR is about 0.6 khp.
SClnc:nptII:SC3Tr
is about 1.7 khp.
FIGURE 19a-c are diagramatic representations of the construction of plasmid
pKHAN4
from pKHAN2 and pKSB.barl. pKHAN4: A HindIII-EcoRl segment containing S7nc
(572 bp), nptII coding region (978 bp) and vicillin 3' end (276bp) from pKHAN2
was
inserted into binary plasmid pKSB.barl to yield pKHAN4; pKHAN2. The nptII
coding
region (978bp, BamHI-Smal fragment) from p35SKN was cloned into Asp718 site
(blunted with klenow fragment) of pKHANl to create pKFiAN 2. SCSV Pro = S7nc;
pKSB. Bar 1: pTABIO.MCSorilB digested with EcoRl as ligated together.
FIGURE 20 is a photographic representation of a selection of Kananmycin
resistant
tobacco plants on regeneration medium transformed with binary vectors
containing either
35S promoter:NPTII:35S terminator sequence (35SPrm:NPTII:35STrm) or
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Slnc:NPTII:SC3Tr expression cassette. (In the Figures, the abbreviations are
35S Prm
NPTII35STrm and SC1 Prm NPTII Sc3 Trm, respectively.
FIGURE 21 is a diagramatic representation of a cloning vector utilising SCSV
DNA
transcription regulatory signals.
FIGURE 22 is a diagramatic representation of SCSV segment 2 dimer construct
pBS2.
This construct was created by cloning a tandem repeat of the SCSV segment 2
DNA
(containing a whole functional seg 2 transcription unit) into the polylinker
site of
pGEM7 which was then cloned into a reduced version of the pMCP3 binary vector
(Khan et al., 1994).
A summary of the transcription activities facilitated by the SCSV
transcription regulatory
elements is shown in Table 19.
EXAMPLE 1
SCSV DNA sequence determination
The F isolate of SCSV (Chu et al. 1993a) was used for sequence determination.
Full-length clones of the SCSV genome components were created from the
replicative
form (RF) DNA as described by Chu et al. (1993a). Other clones were created
from the
RF by PCR with SCSV specific primers designed from known sequences.
The dideoxy chain termination method (Sanger et al., 1977) was used to
sequence either
M13 ssDNA templates (Sambrook et al., 1989) or dsDNA templates prepared by the
CTAB method (Del Sal et al., 1989) from clones described above. Sequence
analysis
was carried out using the University of Wisconsin Genetics Computer Group
Sequence
Analysis Software Package (Devereaux et al., 1984).
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EXAMPLE 2
s Plasmid construction
DNA manipulation techniques used were as described by Sambrook et al. (1989).
The
entire non-coding regions of all 7 DNA segments of SCSV were amplified by PCR
using specific primers (See Table 8) with appropriate restriction enzyme
recognition sites
for cloning into the plasmid vectors. The amplified DNA fragments were
separately
cloned into the respective expression vectors upstream of a promoterless GUS
reporter
gene in the appropriate orientation to produce the SCSV promoter-GUS fusion
constructs
as shown in the Figures 3 and 4. The expression vector pHW9 (Fig. 3) followed
by
cloning into the binary vector pGA470 (An et al., 1985) were used for tobacco
transformation. The expression vector pKGO was used for GUS expression in
protoplasts (Fig. 4) and the binary vector pTAB 10 was used for subterranean
clover
transformation (Fig. 5). pKGO was constructed by cloning the Xhol fragment of
1 S pKIWI l O1 (Janssen and Gardner, 1989) containing the GUS gene and the OCS
terminator sequence into the SaII site of pJKKm (Kirschman and Cramer, 1988).
Corresponding plasmids containing the 35S promoter fused to GUS were used as
controls. The junctions of the clones were checked by sequencing. A
promoterless
GUS construct was used as a control.
Deletion derivatives of the SCSV segments S and 7 promoters were made by
digesting
the full-length non-coding sequence with an appropriate enzyme to produce the
desired
deletion (Fig. 4).
To investigate the phenomenon of transactivadon, the SSnc:GUS expression
cassette was
excised from recombinant pKGO constructs and cloned into a pGEM plasmid
containing
the seg 2 RAP coding region expressed from the 35S promoter. The resultant
plasmid
carrying both the SCSV promoter:GUS and the 35S promoteraeg 2 RAP expression
cassettes were electroporated into protoplasts.
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EXAMPLE 3
Protoplast isolation and transient gene expression
Recombinant plasmids were extracted from E. coli by alkaline lysis followed by
purification through Qiagen columns as described in the Qiagen Plasmid
Handbook.
Suspension cell cultures were used to isolate Nicotiana plumbaginifolia (Last
et al.
1991 ) and subterranean clover cv. Woogenellup protoplasts (Chu et al. 1993b).
Purified
plasmids were electroporated into protoplasts of subterranean clover or
Nicotiana
plumbagin folia as described by Taylor and Larkin ( 1988). Protoplasts were
harvested
3 days later and assayed fluorometrically for transient GUS activity using 4-
methyl
umbelliferyl ~3-glucuronide as substrate (see below). A11 experiments were
done using
duplicate samples per treatment.
EXAMPLE 4
Transformation of tobacco with SCSV-promoter-GUS fusion constructs
The recombinant pGA470 binary constructs containing the various SCSV
promoter:GUS
expression cassettes were separately transformed into Agrobacterium
tumefaciens strain
LBA4404 (Hoekema et al. 1983) by eiectroporation as described by Nagel et al.
(1990).
Nicotiana tobacum cv. Wisconsin 38 were transformed and regenerated as
described by
Ellis et al. (1987).
EXAMPLE 5
Transformation of subterranean clover with SCSV-promoter-GUS fusion construct
The recombinant pTABlO binary vector containing the S7nc:GUS fusion construct
(pBS150) was transformed into Agrobacterium tumefaciens strain AGL1 (Lazo et
al.
1991) by triparental mating (Ditty et al. 1980). Subterranean clover cv.
Larisa was
transformed by Agrobacterium-mediated transformation and regenerated as
described by
Khan et al. (1994).
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EXAMPLE 6
GUS assays
Protoplasts were lysed by sonication in the presence of 0.3% v/v Triton X-100
immediately after harvest. GUS activiri~ of the soluble extract was determined
in a
fluorometric assay using the substrate 4-methyl u.mbelliferyl ~i-D-glucuronide
(MUG)
(Jefferson et al., 1987). Fluorescence was measured using a Labsystem
Fluoroskan II
spectrophotometer.
GUS expression in intact transgenic tissues was detected by histochemical
staining in 5-
bromo-4-chloro-3-indolyl-(3-glucuronic acid (X-gluc) (Jefferson et al., 1987)
and by
fluorometric assay of the soluble extract using MUG.
EXAMPLE 7
Sequence of SCSV non-coding region
The complete sequences of all the 7 known SCSV DNA circles have been
determined
(Fig. 6). Each DNA contains a non-coding region with signals (TATA boxes) for
promoter activity (Table 2). These sequences have not been described or
isolated before.
Sequence comparison of the non-coding regions comprising these promoters
showed that
segments 3 and 5 are most similar, sharing 258 conserved nucleotides out of an
average
of 491 nucleotides in the non-coding regions of the DNAs. Segments 3, 4, 5 and
7
contain 170 conserved bases between them while only 152 bases are conserved
between
Segments 1, 3, 4, 5 and 7. The sequence variations in the non-coding regions
of the
SCSV DNAs suggest that the transcription and replication of the different SCSV
genes
may be regulated differently.
EXAMPLE 8
Transient activity of SCSV promoters in protoplast~
The promoter activities of two SCSV DNA non-coding regions have been
demonstrated
directly by transient expression of GUS using SCSV promoter:GUS fusion
constructs in
subterranean clover (Table 3) and tobacco protoplasts (Table 4). These results
showed
that both segment 5 and segment 7 promoters are functional in the absence of
other
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SCSV DNA components. The SCSV promoters are also functional in protoplasts of
either a natural host (subterranean clover) or a non host (tobacco). In
tobacco
protoplasts, the segment 7 promoter was similar in activity to the 35S
promoter while
the segment 5 (coat protein) promoter consistently showed activity about half
that of the
CaMV 35S promoter (Table 4). However, the activity of both promoters were
higher
in subterranean clover protoplasts, with the activity of the segment 7
promoter showing
up to several times that of the 35S promoter (Table 3), suggesting that SCSV
promoters
work better in certain legumes than the widely used 35S promoter. The activity
of the
segment 7 promoter also appeared to be more variable in subterranean clover
protoplasts
than the others tested.
Plasmids containing various deletion derivatives of the non-coding sequence of
segments
5 and 7 fused to GUS were also constructed (Fig. 4) and electroporated into
protoplasts
(Table 5). GUS assays of protoplasts transfected with these constructs showed
that
neither the stem-loop nor the common region were necessary for promoter
activity
although the latter was required for full activity (Table 5). The DNA sequence
required
for high level promoter activity appears to be less than 300 by which is
smaller than that
required for other promoters, such as 35S promoter (Odell et al., 1985).
EXAMPLE 9
Transactivation of SCSV promoter activity by SCSV segment 2 gene product
When co-electroporated with a 35S promoteraeg 2 RAP gene construct, the
activities
of GUS driven by the seg 5 promoter apparently increased by about 2 fold in
both
subterranean clover and tobacco protoplasts (Table 6). The segment 7 promoter
activity
may also be increased when co-electroporated with the 35Saeg 2 RAP construct
but
further experiments are needed to confirm this.
Transactivation of GUS activity was also apparently observed when the SSnc:GUS
construct was co-electroporated with a binary vector plasmid (pBS2) containing
a
tandem repeat (diner) of the segment 2 DNA (Table 6). A map of SCSV segment 2
diner construct -pBS2 is shown in Figure 22. This construct was created by
cloning a
tandem repeat of the SCSV segment 2 DNA (containing a whole functional seg 2
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transcription unit) into the polylinker site of pGEM7 which was then cloned
into a
reduced version of the pMCP3 binary vector (Khan et al., 1994). The results
suggest
that both SCSV promoters are expressed concurrently. Thus, different SCSV
promoters
can either be used in combination with a 35S promoter or be used
simultaneously to
S facilitate concurrent expression of multiple transgenes in plants. In
contrast, reduced
transgene activity has been observed when the 35S promoter is used in multiple
or
multiple copy transgenes (Line et al., 1990; Matzke and Matzke, 1991; Scheid
et al.,
1991; Carvalho et al., 1992).
EXAMPLE 10
Activity of SCSV promoters in transgenic plants
All the SCSV promoters, except those from segments 2 and 6, have been shown to
be
capable of driving expression of transgenes in tobacco plants (Fig. 7). The
level of
activity of the promoters measured by the MUG assay varied from plant to plant
and
from one promoter to another but is generally lower than that of the 35S
promoter
(Table 7).
Histochemical staining of intact transgenic tobacco tissues showed that the
activity of
the S4nc promoter appears to be constitutive and GUS activity was detected in
all plant
organs. The others are generally restricted to the vascular tissues although
expression
is also detected in pollen.
In transgenic subterranean clover plants expressing the S7nc:GUS gene
construct,
histochemical staining showed that GUS activity is found in leaves, stems and
petioles.
The distribution of GUS activity is mostly constitutive although in some
tissues, the
activity is largely in vascular tissues (Figure 7). In these plants, the level
of promoter
activity also varies from plant to plant but the activity is generally
comparable to that
of the 35S promoter.
These results indicate that the SCSV promoters provide a choice of promoters
that can
be used either independently or simultaneously to control the expression of
one or more
foreign genes in a wide range of plants and tissue types. Legumes are a major
target
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for application. The activity of these promoters also can be enhanced by the
presence
of the seg 2 gene product. Thus, these promoters appear to have significant
advantages
over the CaMV 35S promoter, both in levels of expression, size and in
overcoming some
negative features of the 35S promoter. They would be applicable in a wide
range of
transgenic applications.
TABLE 1
Properties of plant viruses with small and multiple encapsidated circular
ssDNAs
(Plant Circoviruses)
Coat
No. of protean
Virus Hosts Vector circles Size (kb) (kDa)
FBNYV Legumes Aphids 2+ 1 22
MDV Legumes Aphids 1+ 1 21
SCSV Legumes Aphids 7 1 19
BBTV Monocots Aphids 6+ 1-1.2 20
CFDV Monocots Planthopper 1+ 1.3 25
Compiled from data presented at the Sixth International Congress of Plant
Pathology,
Montreal, July, 1993. FBNYV = faba bean necrotic yellow virus, MDV = milk
vetch
dwarf virus, SCSV = subterranean clover stunt virus, BBTV = banana bunchy top
virus,
CFDV = coconut foliar decay virus.
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TABLE 3
SCSV promoter directed GUS activity in subterranean clover protoplasts
GUS Activities
Treatments Expt 1 Expt 2 Avaage
Act %35S Act %35S %35S
20
No DNA 10 0% 13.1 0% 0%
35S:GUS 27.3 100% 36.6 100% 100%
SSnc:GUS 22.6 73% 40.2 116% 95%
S7nc:GUS 36 147% 117 385% 266%
All experiments were done using duplicate samples per treatment. GUS activity
was
measured using a Labsystem Fluoroslcan II spectrophotometer and is presented
both in
absolute activity (Act) and as a percentage of 35S:GUS activity (%35S).
' constructs are represented as "promoter:reporter gene". For example,
"35S:GUS"
is the 35S promoter and the GUS reporter gene. "SSnc" and "S7nc" are tire
promoters for segments 5 and 7, respectively of SCSV.
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TABLE 4
SCSV promoter directed GUS activity in tobacco protoplasts
GUS Activities
Treatments Expt 1 Expt 2 Average
Act %35S Act %35S %35S
No DNA 13.8 0% 10.5 0% 0%
35S:GUS 113 100% 117 100% 100%
SSnc:GUS 52.3 39% 61 48% 44%
S7nc:GUS 93 80% 106 90% 85%
All experiments were done using duplicate samples per treatment. GUS activity
was
measured using a Labsystem Fluoroskan II spectrophotometer and is presented
both in
absolute activity (Act) and as a percentage of 35S:GUS activity (%35S).
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TABLE 5
Promoter activities (GUS expression levels) of deletion derivatives
of segment 5 and 7 non-coding regions in protoplasts.
Levels are expressed as percentabes of the activity of the respective
full-length non-coding sequence.
GUS Activity
Tobacco Subclover
Promoter Deletion$ Expt 1 Expt 2 Expt 1 Expt 2
Segment 5 dNde 101 110 - -
dPml (stem-loop) 65 88 - -
dAfl (Stem-loop+ 70 102 63 91
common region)
dPst S 18 6 4
Segment 7 dAfl (stem-loop+50 55 67 39
common region)
See Figure 4 for maps of deletion derivatives.
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TABLE 6
Transactivation of segment 5 promoter activity (GUS expression)
in tobacco and subterranean clover protoplasts by gene product of segment 2.
GUS Activitya
Promoter ConstructProtoplastsExpt 1 Expt 2 Expt 3 Expt 4
SSnc:GUS Tobacco 100 100 - -
SSnc:GUS + Tobacco 262 217 - -
35S:Seg 2 RAP
SSnc:GUS Subclover 100 100 100 100
SSnc:GUS + Subclover 132 176 188 770
35S:Seg 2 RAP
SSnc:GUS + Subclover - - - 320
Seg 2 dimerb
GUS activities are expressed as percentages of the SSnc:GUS construct in each
experiment
See Example 9
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TABLE 7
Relat~~.~P GUS activity in leaf extracts of independent transgenic
tobacco plants containing different promoter:GUS constructs
as determined by fluorometric assaysa
Promoter GUS Activity Average
Non transgenic control123
Average 123
35S #3 38,431
#6 20,052
#8 20,648
#11 28.325
#12 24,700
Average 26,431
S4nc #1 7,229
#2 4,690
#SB 4,300
#9 6,218
#18 4,895
#19 6,098
Average 5,572
S7nc #4 642
Average (~2
GUS activity presented is the best 25% of the transgenic plants tested.
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EXAMPLE 11
Further characterisation of SCSV promoter activities in transgenic Plants
Transgenic plants of tobacco transformed with the five (Segments 1, 3, 4, 5
and 7)
SCSV promoter:GUS fusion cassettes were assayed for GUS activity by both
histochemical (Figs. 8 and 9) and fluorometric assays (Fig. 10). Samples taken
from
tissue-cultured and young glasshouse-grown plants produced the same GUS
expression
pattern. GUS activity was observed in all plant parts, including roots, stems,
leaves,
petioles and all flower parts. Promoter 5 construct gave relative lower GUS
expression in pollen than other promoters. The results from fluorometric
assays
confirmed previous data showing that segment 4 promoter was the highest
expressor,
with activity 10-fold or greater than the rest (Fig. 10), but is still lower
than that of
the 35S promoter. The expression levels of the segment 1, 3, 5 and 7 promoters
were
comparable to those of the phloem-specific promoter rolC in tobacco
(Schmulling et
al., 1989; Sugaya et al., 1989). Plants transformed with the promoterless GUS
construct did not express GUS by either assay method. Histochemical assays
showed
that expression of all promoter constructs was the highest in vascular
tissues, with high
expressors being more constitutive than low expressors which are more vascular-
limited (Fig. 8). In general, promoter 1, 3, 5 and 7 constructs are expressed
mostly in
the vascular tissues. In particular, GUS expression by promoter 1 and 3
constructs are
mainly restricted to phloem tissues. However, for all promoters histochemical
staining
of leaves showed that GUS expression in these tissues are often blotchy
(constitutive
and vascular-limited) and variable between leaves of the same plant. Dark
field
microscopy (Jacobsen-Lyon et al., 1995) also showed that none are strictly
vascular-
limited (Fig. 9).
Twenty primary transgenic subterranean clover plants expressing the seg 7
promoter:GUS gene were further characterised by histochemical assays (Table
9).
These assays showed that GUS activity was observed in all plant parts,
including
roots, stems, leaves and petioles. GUS expression was the highest in vascular
tissues,
with some leaves being more constitutive and blotchy than other organs and
high GUS
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expressing plants being more constitutive than low expressing ones. Samples
taken
from tissue-cultured and glasshouse-grown plants produced the same GUS
expression
pattern.
EXAMPLE 12
Detection of promoter activity in SCSV segment 2 DNA
Experiments showed that the non-coding regions from the SCSV segments 2 and 6
DNAs were unable to drive the expression of GUS gene in transgenic tobacco.
These
regions are only 179 and 159 nucleotides long, respectively, and it is likely
that
additional sequences are required for promoter activity. To test this
hypothesis, a new
segment 2 promoter sequence was constructed consisting of the DNA fragment
from
nucleotides 526 to 46 and fused to the promoterless GUS vector pKGO (Fig. 11
). The
fusion construct was electroporated into tobacco protoplasts. Gus activity was
detected
in electroporated tobacco protoplasts at levels similar to that of segment 5
promoter:GUS construct (Table 10).
A binary vector containing the above SRnc promoter:GUS fusion DNA was also
transformed into tobacco plants as described in Example 4. Histochemical
staining of
several transformed tobacco plants showed that GUS expression was mainly
vascular.
These results showed that additional sequence from the SCSV segment 2 DNA
coding
region is necessary for promoter function. Since the SCSV segment 6 is a
variant of
the segment 2, it is expected that a similar construct comprising the
noncoding and
part of the coding region of this DNA will produce an active promoter. Thus,
all
SCSV promoters are expected to be suitable for driving gene expression in
plants.
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EXAMPLE 13
Enhancement of gene expression by SCSV transcription termination and
polyadenylation signals
Effective gene expression requires not only a promoter, but also specific
nucleotide
sequences at the 3' end of the coding region of the gene, known as the
termination and
polyadenylation signals (Messing et al., 1983; Joshi 1987b; Gil and Proudfoot,
1984;
Rothnie et al., 1994). These special sequences are required to signal the RNA
polymerase to stop transcription and to allow further processing of the RNA
transcript.
The activity of the termination and polyadenylation signals may affect the
transcription
efficiency and stability of the RNA being transcribed. Some widely used
termination/polyadenylation sequences include those from the NOS (Depicker et
al.,
1982), OCS (Janssen and Gardner, 1989) and CaMV 35S (Pietrzak et al., 1986)
genes.
Each of the SCSV DNA segment (or component) contained a terminator sequence
comprising a termination and a polyadenylation signal sequence in the
noncoding
region (Table 11; Figure 2). To demonstrate activity of the
termination/polyadenylation signals in SCSV DNA, GUS expression vectors
containing
either the segment 3 or segment 5 polyadenylation and termination signal (Fig.
12)
was constructed and subjected to transient expression in tobacco protoplasts.
The
respective terminator sequence was amplified by PCR with several restriction
sites
incorporated into the primers (Fig. 12). The amplified terminator fragments
were cut
with the indicated restriction enzymes and cloned into the pKGO recombinant
plasmid
containing either Slnc or S4nc promoter:GUS:OCS3' constructs, from which the
OCS3' sequence has been deleted (Fig. 12). The resultant Slnc:GUS:SC3Tr (the
segment 1 promoter here carries a deletion of the XindIII fragment from
nucleotides
641-782 which has no effect on GUS activity) and S4nc:GUS:SSTr constructs were
electroporated into tobacco protoplasts and assayed for GUS activity. The
results
showed that GUS activity was increased 2 to 3-fold when the SCSV
tenmination/polyadenylation sequence was used instead of the commonly used OCS
termination/polyadenylation sequence in the same construct (Table 12). In the
same
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experiment, the construct Slnc:GUS:S3Tr produced over two-fold higher activity
than
the 35S:GUS:OCS3' construct (Table 12). These results indicate that each of
the
SCSV DNA components contains a different termination and polyadenylation
signal
sequence which can be used in various combination with the SCSV promoters to
regulate and/or enhance expression of foreign genes in plants. As with the
SCSV
promoter sequences, the SCSV termination/polyadenylation sequences are
advantageous over currently available termination/polyadenylation sequences by
their
small sizes (160-170 nucleotides) and the availability of a broad range of
transcription
regulators with different strengths and tissue specificities for genetic
manipulation.
The results also show that the use of a SCSV promoter in combination with a
SCSV
terminator sequence in higher levels of gene expression than constructs using
the 35S
promoter in conjunction with one of the common transcription terminator
sequences.
EXAMPLE 14
Activity of SCSV promoters in transgenic potato plants
pGA470 binary vector containing the S4nc:GUS:NOS fusion construct cloned in
pHW9 (Figure 3) was used to transform potato plants. pHW9 is derived from pHW8
(Dolferus et al., 1994) into which the polylinker from pGEM3zf(+) is inserted.
The
recombinant binary vector was transformed into Agrobacterium tumefaciens
strain
LBA4404 by electroporation (Nagel et al., 1990). Potato cultivars Atlantic and
Sebago were transformed and regenerated essentially as described by Wenzler et
al.
(1989) except for the following modifications. Stem pieces instead of leaf
pieces were
used for transformation and 10 mg/1 of benzylaminopurine (BAP) instead of 2.24
mg/1
was used during co-cultivation. After co-cultivation, stage 1 medium is
supplemented
with 100mg/1 of cefotaxime and not kanamycin or carbenicillin. Stage II medium
contained 2 mg/1 BAP, 5 mg/1 GA3, 100 mg/1 kanamycin and 100 mg/1 cefotaxime.
Six transformed plants comprising 5 of cultivar Atlantic and 1 of cultivar
Sebago were
transferred and grown in the glasshouse for 10-11 weekswntil small tubers
formed.
Tissues from different parts of the plants were assayed by histochemical GUS
staining.
The results showed that GUS was highly expressed in all plant parts including
roots,
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stolons, tubers, stems and leaves but the expression was predominantly
vascular,
including cambium, phloem elements and some xylem elements (Fig. 13). As in
other
transgenic hosts, GUS expression in non-vascular tissues of highly GUS active
plant
materials, especially young tubers, was more evident than in less active
tissues when
compared with plants transformed with a 35S:GUS:NOS construct, SCSV promoter
directed GUS expression in tubers was at least as high as that of the 35S:GUS
construct.
EXAMPLE 15
Activity of SCSV segment 7 promoter in transgenic cotton plants
pGA470 binary vector containing S7nc:GUS:NOS fusion construct cloned in pHW9
was used to transform cotton plants. The recombinant binary vector was
transformed
into Agrobacterium tumefaciens strain AGL 1 by triparental mating. Cotton
(Gossypium hirsutum) cv. coker 315 was transformed and regenerated as
described by
Cousins et al. ( 1991 ).
Transformed plants were grown in the glasshouse and leaf tissues from 18
independent
transgenic plants were assayed for GUS activity by histochemical staining. GUS
activity varied between plants. Five of these plants showed strong GUS
expression,
similar in range to 35S promoter driven GUS expression and was predominantly
in the
vascular tissues (Fig. 14). GUS activity was especially strong in the gossypol
glands.
As in other transgenic hosts, GUS staining in highly expressed tissues were
also
constitutive.
A variety of tissues from these plants were then stained and vascular
expression was
observed in all organs including roots, stems, petioles, petals and other
vascularised
floral parts. Expression appears to be particularly high in young flower buds.
Seedlings from one of the lines was screened for GUS activity. All 10
progenies
stained heavily in roots and leaves indicating that the gene was inherited and
that the
line probably contained more than one independent insertion site.
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EXAMPLE 16
Stability of transformed SCSV promoter:GUS expression cassette in transgenic
plants
S The stability of GUS expression driven by the various SCSV promoters in
transgenic
tobacco and subclover were further characterised in T1 generation seedlings of
subterranean clover and tobacco plants.
In tobacco, T1 seedlings from 10 independent transgenic lines were assayed by
histochemical staining. The results showed that the expression of the GUS gene
driven by all the five (segments 1,3,4,5 and 7) promoters was stable in the T1
seedlings, with the pattern of expression being maintained in all cases
between TO and
T1 plant tissues of the same age. In very young stems where the vascular
tissues are
not well differentiated, expression from all promoters were very high and was
detected
through out the stem tissues. Gradual vascular limitation occurs with age and
with
increasing differentiation of the vascular bundles. As with TO plants, the
segment 4
promoter mediated GUS expression was more constitutive than others.
Fifty 2-3 month old T1 seedlings from 15 independent transgenic subclover
plants
expressing the S7nc:GUS fusion construct were assayed for GUS activity by
histochemical and fluorometric assays. The results showed that the expression
of the
GUS gene driven by the segment 7 promoter was stable in T1 seedlings, with the
pattern of expression being maintained between TO and T1 plant tissues of the
same
age. GUS expression was found to generally segregate at the expected ratio of
3:1.
The preliminary results from, fluorometric assays confirmed the histochemical
data
suggesting that this segment 7 promoter construct had GUS activity somewhat
lower
than that of the 35S promoter in leaves and petioles (Table 13). GUS activity
in
subterranean clover stems, however, was 3-fold higher than in petioles and 6-
fold
higher than in leaves (Table 14). The age of the plants at the time of assay
was 2 to 3
months.
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EXAMPLE 17
Transient activity of SCSV promoter in soybean leaves
The SCSV promoter:GUS construct used (Table 15) was derived from the
promoterless GUS plasmid pKGO (Figure 4; pJKKmf(-) K1W1 GUS:OCS) described
previously while the 35S promoter:GUS construct was pGUS. pGUS is derived by
cloning the Gus gene from pKIWI101 into the plant expression vector pDH51
(Pietrzak et al., 1986).
For transient GUS expression in soybean tissues, the GUS constructs were
introduced
into tissues tissues by particle bombardment using the Bio-Rad Biolistic PDS-
1000/He
Particle Delivery System as above. A 501 suspension containing 3mg of a 1:1
ratio
of 1 and 5~ gold particles plus hug of DNA was shot onto plates containing 3
leaves
each. Fully expanded leaves used in these experiments were prepared from 24
day old
soybean plants cv. Wayne. After particle bombardment, GUS activity was assayed
24
hours later by vaccuum infiltration of the leaves with X-Gluc (Craig, 1992).
The results (Table 15) showed that in transient expression in soybean leaves,
SCSV
segment 4 promoter was more active (25-35 spots/leaf) than the 35S promoter
(10-15
spots/leaf) when the respective plasmids were shot into soybean leaves.
EXAMPLE 18
Testing of SCSV promoters for callus-specific expression
All seven SCSV non-coding sequences were cloned into the promoterless GUS
vector
pHW9. Binary vectors each containing one of the seven SCSV promoter:GUS fusion
constructs were transformed into tobacco tissues by Agrobacterium-mediated
gene
transfer. At 2-3 weeks after transformation, calli containing primodia of
transformed
shoots were subjected to histochemical GUS staining. Best expression was
observed
in calli transformed with segment 1 followed by the S4nc:GUS:OCS constructs.
This
result suggests that the segment 1 promoter is best suitable for selectable
marker gene
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expression and that the segment 4 promoter is best for gene expression.
EXAMPLE 19
Characterisation of regulatory sequences of the Flaveria bidentis MeA gene
In Flaveria bidentis (Chitty et al., 1994) the MeA gene is the gene that codes
for the
NADP-malic enzyme adapted for C4 photosynthesis. The structure and putative
promoter and transcription termination/polyadenylation signal sequences of
this gene
has been isolated and the terminator sequence determined (Fig. 15). The
potential
activities of the putative promoter element (MeA 5' sequences [MeA 5'] and the
terminator MeA 3' sequences [MeA 3']) of the F. bidentis MeA gene were studied
in
transgenic F. bidentis plants using GUS expression vectors (Fig. 16). A long
version
of the MeA 3' terminator sequence (MeA 3'L = S.Skb from the stop codon) was
used
in these experiments. In Fig. 16, the GUS expression cassette ME20 is ligated
to the
binary vector pGA470 (An et al., 1985) while ME29 is cloned into pGA482 (An,
1986). Plants were transformed with these vectors as described by Chitty et
al. ( 1994)
Study of GUS activity of the transformed plants by histochemical staining and
fluorometric assays showed that the MeA 3' sequence of the gene is required
for high
level expression of GUS in leaves of transgenic F. bidentis plants (Table 16).
The 5'
sequences of the gene do not appear to contribute to gene expression in leaves
but
appear to direct expression in meristems and stems in the presence of a
suitable
transcription termination/polyadenylation signal sequence such as the OCS 3'
(Table
16).
EXAMPLE 20
Use of the MeA 3' termination/polyadenylation signal sequences in SCSV
promoter constructs to enhance gene expression in monocotyledenous plants
Because most gene control elements are located at the 5' end, the activity of
the MeA
3' sequence is tested in conjunction with the S4nc SCSV promoter with the view
to
enhance gene expression directed by the SCSV promoters. For these experiments,
a
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66718-18
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short version of the MeA 3' terminator sequence was used (MeA 3'S; 900 bases
from
the stop codon) to prepare GUS expression vectors containing the S4ne promoter
with
either the OCS 3', SCSV segment 5 3' (SCSTr) or the MeA 3' transcription
termination/polyader_ylation signal sequence. These constructs were derived
from the
promoterless GUS plasmid pKGO described previously and the recombinant plasmid
pBS237 containing the S4nc:GUS:MeA3' construct is presented in Fig. 17. GUS
activity conferred by these constructs were assayed in Japonica rice callus
cv. Taipei
309. The constructs were introduced into rice calli by particle bombardment
using the
Bio-Rad Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories)
and
compared with results obtained in dicotyledonous tissues such as soybean
leaves and
tobacco protoplasts (Table 17). For rice particle bombardment experiments, 4
mg of a
1:1 ratio of 1 and S micron gold particles plus 5 ug of DNA in a total of SOuI
volume
was shot onto six plates of calli. The DNA was made up with a 4:1 molar ratio
of
each of the vectors containing GUS gene to the vector containing the
selectable
marker gene. The vector containing the selectable marker was pTRA 151 (Zheng
et
al., 1991). Each plate contained 50-100 fresh secondary calli derived from
mature
embryos. Forty hours after DNA bombardment, GUS activity was detected by
placing
the calli in 0.3% w/v X-Gluc solution in 100 mM phosphate buffer. Blue spots
were
counted after overnight incubation.
For transient GUS expression in tobacco and soybean, only the GUS constructs
were
introduced into protoplasts and leaf tissues, respectively. After
electroporation,
tobacco protoplasLs were assayed for GUS activity as previously described. The
constructs were introduced into soybean tissues by particle bombardment as
described
above.
The results showed that in the monocotyledenous rice tissues, a 16-fold higher
activity
was obtained with the MeA 3' construct compared to the SCSV terminator (Table
17).
In similar experiments, a highly expressed GUS construct containing the
ubiquitin
promoter:GUS:NOS cassette (Christensen et al., 1992) has about 4-fold higher
activity
than the SC4:GUS:MeA3' construct.
*Trade-mark
WO 96!06932 PCT/AU95/00552
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In the dicotyledonous tissues, similar activities were obtained with both of
these
constructs in tobacco protoplasts and soybean leaves. However, both were 2-
fold
higher in activity than that obtained with the OCS terminator in tobacco
protoplasts
(Table 17).
These results suggest that the MeA3' , sequence can be used to enable gene
expression
directed by SCSV promoters in monocots.
EXAMPLE 21
Use of new vectors containing SCSV promoters and terminators to drive a
selectable marker gene in transgenic plants
The suitability of using SCSV promoters to drive a selectable marker gene as a
basis
for selecting transgenic plants after transformation and regeneration was
tested in
tobacco plants. The selectable marker used is the kanamycin resistance gene,
nptII.
Binary vectors containing either a SCSV segment 1 (pBS246) (Fig. 18) or a SCSV
seg
7 promoter (pKHAN4) (Fig. 19) fused to the nptII gene were constructed from
the
pART27 (Gleave, 1992) and pKSB.barl (Figure 19), respectively. These were
transformed separately into tobacco plants (Ellis et al., 1987) and putative
transgenic
plants were selected under kanamycin selection using 100 pg/ml kanamycin (Fig.
20).
Kanamycin resistance was confirmed in the transgenic plants by dot blot assay
for the
nptII gene activity (McDonnell et al., 1987) and survival of the transgenic
plants
under 100pg/ml kanamycin in a rooting medium. The results showed that the SCSV
segment promoter construct produced at least as many kanamycin resistant
plants as
the 35S promoter construct use in the same experiment and is, therefore, as
effective
as the 35S promoter for selecting transgenic tobacco plants based on kanamycin
resistance (Table 18). Tobacco transformed with pKHAN4 is resistant to 100
pg/ml
kanamycin in regeneration medium and 50 pg/ml kanamycin in rooting medium.
Restriction maps of pKSB.barl and pKHAN2 used to produce pKHAN4 are shown in
Figure 19.
SUBSTITUTE SHEET (Rule 26)
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EXAMPLE 22
Development of a new plant gene expression vector system
A new expression vector comprising a SCSV segment 4 promoter and a SCSV
segment 5 terminator driving any useful gene of interest (pICAN 1 ) (Fig. 21 )
has been
constructed from a pGEM derivative and the resultant expression cassette can
be
inserted into the binary vectors pBS246 and pKHAN4. The resultant binary
vectors
can then be used to transform plants of economic importance especially cotton,
subclover, potato and white clover under kanamycin selection. Other binary
vectors
can be constructed from different combinations of SCSV promoters and
terminators to
produce a full range of binary vector system for plant gene expression.
SUBSTITUTE SaEET (Rule 26)
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TABLE 9
GUS activity of SCSV segment 7 promoter:GUS construct in primary
transgenic substerranean clover plants (To)
Plant # Basta Resistance GUS Activity
1 R +++
2 R ++
3 R -
4 R ++
5 S ++
6 R -
7 R ++++
8 R ++++
1 9 MR +++
S
10 R +++
11 R +++
12 MR -
13 MR ++
14 S ++
15 R +
16 N.d. ++++
17 N.d. +++
18 N.d.
19 N.d. +++
20 N.d. ++
Plants were analysed two months after transfer of plants to glasshouse from
tissue culture. Banta (phosphinothricin - [PPT] resistance was assayed by
painting basta at 1 gm PPT/litre onto fully unfolded young leaves and reaction
was assayed after one week.
R No damage;
MR Moderate damage to
leaflet;
S Leaflet dead
N.d. Not determined
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TABLE 10
GUS activity in tobacco protoplasts directed by SCSV segment 2 promoter
to tobacco protoplasts relative to the 35S promoter
Construct GUS Activity
Experiment
1 2
S2nc:GUS:OCS3' 0.46 0.45
35S:GUS:OCS3' 1 1
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TABLE 11
Putative Polyadenylation and Termination signals in SCSV DNA Components
DNA Component Putative Polyadenylation/Termination
Signals
Seg.l AATTAT 19 TGTGTTTT
Seg.2 AATAAA 10 TTGTTTT
Seg.3 AATAAA 3 TTGTT
Seg.4 AATAAA 8 TTATTGTT
Seg.S AATAAA 3 TTGTTTT
Seg.6 AATAAA 9 TTGTT
Seg.7 AATAAA 11 TTGTTT
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TABLE 13
Fluorometric GUS Assay of Independent T1 generation of transgenic subterranean
clover
plants expressing either the S7nc:GUS or the 35S:GUS Construct
Construct/Plant # Leaf (Young) Petiole (Young)
[unfolded] (unfolded]
S7nc:GUS Plant # 1 56.6 120
S7nc:GUS Plant # 2 35 4g
S7nc:GUS Plant # 3 116 140
355:GUS Plant # 1 120 238
355:GUS Plant # 2 280 238
Plants were 2 - 3 months old when assayed.
Results show differential expression in different tissues.
TABLE 14
Distribution of GUS activity in a T1 generation transgenic
subterranean clover plant expressing the S7nc:GUS construct
Source of Tissue Leaf Petiole Stem
Top - [folded leaf; 1 1.9 2.0
# 1 leaf position]
Middle 1.6 5.8 22.0
[#8 leaf position]
Bottom 4.0 4.8 12.6
[# 17 leaf position]
Plants were 2 - 3 months old when assayed.
Results show differential expression in different tissues.
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TABLE 15
Transient expression of GUS in soybean leaves directed by a SCSV promoter
Constructs GUS Expression in
soybean leaves
(# spots/leaf)
S4nc:GUS:SCSTr 25 - 35
35S:GUS:35STr 10 - 15
* Results from one experiment in which spots from 3 leaves were counted in
each
treatment
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TABLE 16
Characterisation of GUS activity directed by MeA 3'L sequence (S.Skb version)
in
Transgenic Flaveria bidentis Plants
Constructs GUS Expression
MeA:GUS:MeA 3'L (ME24) High GUS in leaves
High GUS in meristem
Moderate GUS in stems
MeA:GUS:OCS 3'(ME20) No GUS in leaves
High GUS in meristem
Moderate GUS in stems
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TABLE 17
Transient expression of GUS in rice callus, soybean leaves and tobacco
protoplasts
showing enhancement of SCSV promoter activity by MeA 3's sequences in rice
callus
Constructs GUS Expression
Rice callus Soybean leaves Tobacco protoplasts
(Rel # spots) (Rel # spots)' (Fluorescence)
S4nc:GUS:OCS 3' NDa ND 0.49
S4nc:GUS:SCSTr 0.06 1 1.0
S4nc:GUS:MeA 3'S 1 1 1.1
ND Not done
* Average number of spots from 3 leaves in one experiment
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Hatch, M.D. (1987) C4 photosynthesis: A unique blend of modified biochemistry,
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SUBSTIT'U'TE ST~i:T (P,nle 25)
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Khan, M.RL, Tabe, L.M., Heath, L.C., Spencer, D. and Higgins, T.J.V. (1994).
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-54-
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SUBSTTTUTIr SHE: T (P,ule 26)
42 ~ 9~~ 2~
WO 96/06932 PCT/AU95/00552
-56-
SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL
RESEARCH ORGANISATION
(ii) TITLE OF INVENTION: NOVEL PLANT PROMOTERS AND USES
THEREFOR
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DAVIES COLLISON CAVE
(B) STREET: 1 LITTLE COLLINS STREET
(C) CITY: MELBOURNE
(D) STATE: VICTORIA
(E) COUNTRY: AUSTRALIA
(F) ZIP: 3000
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: AU PROVISIONAL
(B) FILING DATE: 07-NOV-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PM7770/94
(B) FILING DATE: 30-AUG-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HUGHES DR, E JOHN L
(C) REFERENCE/DOCKET NUMBER: EJH/EK
(ix) TELECOh~VIUNICATION INFORMATION:
(A) TELEPHONE: +61 3 9254 2777
(B) TELEFAX: +61 3 9254 2770
~UBSTiTtJTE ;; v:~T ( Rule 2~)
WO 96/06932 ~ ~ ~ PCTlAU95/00552
-57-
(2) INFORMATION FOR SEQ ID NO: l:
. (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1001 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
TAGTATTACC CCCGTGCCGG GATCAGAGAC ATTTGACCAA TAGTTGACTA GTATAATAGC60
CCTTGGATTA AATGACACGT GGACGCTCAG GATCTGTGAT GCTAGTGAAG CGCTTAAGCT120
GAACGAATCT GACGGAAGAG CGTTCACACT TAGATCTAGT TAGCGTACTT AGTACGCGTT180
GTCTTGGGTC TATAAATAGA GTGCTTCTGA ACAGATTGTT CAGAATTTCA TAGCGAGATG240
GATTCTGGTG ATGGTTACAA TACATACTCA TATGAAGAAG GTGCTGGAGA TGCGAAGAAG300
GAAGTTTTAT ATAAAATAGG TATTATTATG TTATGTATTG TAGGGATTGT AGTTTTATGG360
GTTTTAATTA TATTATGTTG TGCTGTTCCT CGCTATGCTA AATCAACGAT GGACGCTTGG420
TTATCTTCGT CTTCTATTAT GAAGAGGAAG ATGGCTTCAA GGATTACTGG TACTCCGTTT480
GAAGAAACTG GTCCTCATCG TGAAAGAAGA TGGGCTGAAA GAAGAACTGA AGCGACGAAC540
CAGAATAATA ATGATAATGT AAATAGATTT AGTTGATATG TTGTAATTTT ATATGGATTA600
ATGAGAATTA TTATTATTCT GTTCTTCGTC TGTGTTTTTT AAGCTTTTTC TGTGTTTTAA660
TGGCGTCTGG AGAGAGAAAG GAATAATTGT AAGGTAGACG ACGATGTAGT GGATTACAGT720
TGTCTTTACT TCGCCTCGAA GAAAGACACA TTTCAAGTTG TGAGTGTTAT TGCTTTTGAG780
GAAGCTTCCT CGAAGCAGCG TATAACTTTA ATTTGAATTT GGTTTTGGCG CGTTAGTGAA840
ATTGCGGCTG TAAACGTGTC AAGTTGTGAG TGGCTGAAAT AAGATAATAG ATATATTATT900
ATTGTTTTAA TTTAATTCCG CGAAGCGATA TGTTAAGTGA TAAATGAAAC GAAGCGTTTT960
GATGACGTCA TATGTCTCCG TGCCTACGTC AGCACGGGGC T 1001
S~ST~TE S~~T (f'.u:e 25)
WO 96106932 ~ ~ ~ pCT/AU95/00552
-58-
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQLTc,ivCE CHARACTERISTICS:
(A) LENGTH: 1022 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
TAGTATTACC CGACCTTGCC ACACCTCCTT GGAACACTTT CTCTCTCTAG60
AAAGTGTGAG
ACTTTCTCTC TCTAAGCTTA TATGGCTAGA AGGTACTGTT TTACATTAAA120
TTACGCTACT
GAGATAGAGA GAGAAACATT CCTCTCCCTC TTCTCTCAAG ACGAATTAAA180
CTATTTCGTT
GTCGGCGACG AAACTGCAAC TACTGGACAG AAACACCTCC AGGGATTTGT240
ATCGTTCAAG
AACAAAATTC GTCTTGGTGG ATTGAAGAAG AAATTTGGTA ATCGAGCTCA300
CTGGGAAATT
GCGAGAGGCA GCGATTCTCA GAATCGCGAT TATTGCTGTA AAGAAACCCT360
AATTTCTGAA
ATTGGGATTC CGGTCATGAA GGGTTCGAAC AAGCGGAAGA CGATGGAGAT420
TTATGAAGAG
GATCCCGAAG AAATGCAATT GAAGGATCCA GATACTGCTC TTCGATGTAA480
GGCGAAGAAA
TTGAAAGAGG AATATTGTTC CTGTTATGAT TTTCAGAAAC TCCGTCCATG540
GCAAATTGAG
CTTCACGAGG ATTTAATGGC GGAACCAGAT GATCGGAGTA TCATCTGGGT600
CTATGGTTCA
GACGGAGGAG AAGGAAAGAC GAGCTTCGCG AAGGAATTAA TCAGGTATGG660
ATGGTTTTAT
ACAGCCGGAG GGAAGACCCA GGACGTATTA TATATGTATG CTCAAGACCC720
AGAGAGGAAT
ATTGCGTTTG ATGTTCCCAG GTGTTCTTCG GAGATGATGA ACTATCAGGC780
GATGGAGATG
TTGAAGAACA GAGTTTTTGC AAGTACAAAA TATAGGCCTG TAGATCTTTG840
TATTAGGAAG
TTAGTTCATT TAATTGTGTT TGCCAACGTG GCACCTGACC CCACGCGCAT900
AAGTGAGGAC
AGACTTGTAA TTATCAATTG TTGAATAAAA GAATATATAT TATTGTTTTA960
ATTTAATTCC
GCGAAGCGGT AGCCGGTCAT AACACTGTTG CCCTTGGAAC ACTATATATA1020
GCAAGGTCGG
CT
1022
st~ss~. ~~ T ~.~~ z~~
WO 96/06932 ~ ~ PCT/AU95/00552
-59-
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 991 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
TAGTATTACC CCCGTGCCGG GATCAGAGAC ATTTGACCAA TAGTTGACTA TGAATAATAG 60
CCCTTGGATT AGATGACACG TGGACGCTCA GGATCTGTGA TGCTAGTGAA GCGCTTAAGC 120
TGAACGAATC TGACGGAAGA GCGGACATAC GCACATGGAT TATGGCCCAC ATGTCTAAAG 180
TGTATCTCTT TACAGCTATA TTGATGTGAC GTAAGATGCT TTACTTCGCC TCGAAGTAAA 240
GTAGGAAATT GCTCGCTAAG TTATTCTTTT CTGAAAGAAA TTAATTTAAT TCTAAATTAA 300
ATTAAATGAG TGGCTATAAA TAGATGTTTC GTCTTCGTTG TTTTACAACG AAGCTTAGAA 360
TCTTGTGTTA ATGGCGTTAA GGTATTTCTC TCATCTTCCT GAAGAACTGA AGGAGAAGAT 420
TATGAACGAG CACTTGAAGG AAATTAAGAA GAAGGAATTT CTAGAGAATG TAATTAAAGC 480
TGCGTGTGCT GTGTTCGAAG GTTTAACAAA GAAGGAGTCT GTTGAAGAAG ACGACATACT 540
ACGCTTCTCT GGGTTTCTGG AAGGTCTGTC TGCATATTAT GCAGAGGCGA CGAAGAAGAA 600
GTGTTTAGTT AGATGGAAGA AGAGCGTTGC AATAAATCTG AAATGGAGAG TTATGGAGGA 660
GATGCATTAC AAGCTTTATG GATTTGCAGA CATGGAAGAT TTATATTATT CAGAGTTAGG 720
GTTTCCTAAT TACGGTGAAG ACGATGTAGC TTATCACGAT GGTGCAATTG TAAATTGTAA 780
GCAATTAGAA GTTGTATTTG ATGATTTAGG TATTGAGTTT ATGTCTATTG TAATTGATAG 840
AGGTTCTATT AAGATAGAAT TATGAGATGT AATTGTGATT AATGAATAAA GAGTTGTTAT 900
TATTCTTTGA ATTACTCCGC GAAGCGGTGT GTTATGTTTT TGTTGGAGAC ATATGACGTC 960
ATATGTCTCG CCGACAGGCT GGCACGGGGC T 991
v7 ~TB~" S.
WO 96/06932 ~ ~ PCT/AU95/00552
-60-
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1002 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
TAGTATTACC CCGTGCCGGG ATCAGAGACA TTTGACTAAA TGTTGACTTG60
GAATAATAGC
CCTTGGATTA GATGACACGT GGACGCTCAG GATCTGTGAT GCTAGTGAAG120
CGCTTAAGCT
GAACGAATCT GACGGAAGAG CGGACAAACG CACATGGACT ATGGCCCACT180
GCTTTATTAA
AGAAGTGAAT GACAGCTGTC TTTGCTTCAA GACGAAGTAA AGAATAGTGG240
AAAACGCGTA
AAGAATAAGC GTACTCAGTA CGCTTCGTGG CTTTATAAAT AGTGCTTCGT300
CTTATTCTTC
GTTGTATCAT CAACGAAGAA GTTAAGCTTT GTTCTGCGTT TTAATGGCGG360
ACTGGTTTCA
CAGTGCGCTT AAGACATGTA CTCATGTCTG TGATTTTTCA GATATTAAGG420
CGTCTTCACA
ACAGGATTTC TTCTGTTGTG ATAGTATGCG AGGTAAATTA TCTGAACCTA480
GGAAGGTGTT
GTTAGTTAGT TGTTTTGTAA GTTTTACTGG TAGTTTTTAT GGAAGTAATA540
GGAATGTTAG
AGGTCAAGTT CAGTTGGGTA TGCAGCAAGA TGATGGCGTT GTTCGTCCAA600
TAGGATATAT
TCCTATTGGG GGTTATTTGT ATCATGATGA TTATGGATAT TATCAAGGAG660
AGAAGACGTT
CAATCTGGAC ATCGAGTCAG ATTATCTGAA GCCTGATGAA GATTTTTGGA720
AGAGATTTAC
AATTAATATT GTAAATGATA AAGGATTAGA TGATAGGTGT GATGTAAAAT780
GTTATGTAGT
TCATACGATG CGTATTAAGG TGTAATTGTT ATTATCAATA AAAGAATTTT840
TATTGTTATT
GTGTTATTTG GTAATTTATG CTTATAAGTA ATTCTATGAT TAATTGTGAA900
TTAATAAGAC
TAATGAGGAT AATAATTGAA TTTGATTAAA TTAACTCTGC GAAGCTATAT960
GTCTTTCACG
TGAGAGTCAC GTGATGTCTC CGCGACAGGC TGGCACGGGG CT 1002
SUBSTITUTE SHEET (RULE 26)
WO 96!06932 ~ ~ pCTlAU95/00552
-61-
(2) INFORMATION i0ic SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 998 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TAGTATTACC CCGTGCCGGG GTCAGAGACA TTTGACTAAA TATTGACTTG GAATAATAGC60
CCTTGGATTA GATGACACGT GGACGCTCAG GATCTGTGAT GCTAGTGAAG CGCTTAAGCT120
GAACGAATCT GACGGAAGAG CGTCATGGTC CACATGTCTA AAGAATAATG CTTTACAGCT180
GTATTGATTT GACTTTACGC GCTTTACTTT AATTGCTTTA AGTAAAGTAA GATGCTTTAC240
TTTGCTCGCG ACGAAGCAAA GTGATTGTAG CTGCAGAAAT TGATGCTTTA ATTACCGGGT300
AACACGGTTT GATTGTGGGT ATAAATATGT TCTGTTCGTT TTCTTCGTTG TCATTTTACA360
ACGAAGATGG TTGCTGTTCG ATGGGGAAGA AAGGGTCTGA GGTCTCAAAG GAGAAAATAT420
.
TCGCGAATTG CTTACAAACC TCCTTCGTCT AAGGTTGTAA GTCATGTGGA GTCTGTTCTG480
AATAAGAGAG ATGTTACTGG AGCGGAGGTT AAGCCATTCG CTGATGGTTC AAGGTATAGT540
ATGAAGAAGG TAATGTTGAT TGCAACATTA ACTATGGCTC CTGGAGAATT AGTTAATTAT600
CTTATTGTGA AGAGTAATTC GCCTATTGCG AATTGGAGTT CGTCTTTCAG TAATCCTTCG660
TTGATGGTGA AAGAGTCTGT TCAAGATACA GTTACGATTG TTGGAGGAGG AAAGCTTGAG720
TCTTCTGGTA CTGCTGGTAA AGATGTAACT AAGTCTTTTA GGAAGTTTGT TAAGCTGGGT780
TCAGGTATTA GTCAGACCCA GCATTTGTAT TTAATTATTT ATTCCAGTGA TGCGATGAAG840
ATCACACTGG AGACGAGAAT GTATATTGAT GTATAATTGT GATGATTAAT GAATAAAGAG900
TTGTTTTTAT TCTTTGAATT ACTCCGCGAA GCGGTGTGTT ATGTTTTTGT TGGAGACATA960
TGACGTCATA TGTCTCCGCG ACAGGCTGGC ACGGGGCT 998
~Ui~S iZTUTE SF~T :,.tI~ Zf)
WO 96/06932 ~ ~ ~ ~ pCT/AU95/00552
-62-
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1017 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CAGTATTACC GCACCTCGCT TACCCTCCTC GCTTCCCTGG GCCCACTATG60
CCTACTAGAC
AAAGCACTAG TTGGGTGTTC ACACTTAACT TTGAGGGCGA AATTCCTATT120
TTGCCCTTTA
ATGAAAGCGT TCAGTACGCT TGTTGGCAGC ATGAGAGAGT GGGACACGAT180
CATTTACAGG
GATTTATACA ATTTAAATCC CGCAACACTA CATTGCGTCA GGCTAAGTAT240
ATTTTTAATG
GACTGAATCC TCATCTGGAA ATTGCTAGGG ATGTAGAGAA GGCGCAATTG300
TACGCGATGA
AGGAAGATAG TAGAGTAGCT GGTCCCTGGG AGTATGGGTT GTTTATTAAG360
AGAGGATCGC
ATAAGCGTAA GCTGATGGAG AGATTTGAAG AAGATGGAGA AGAGATGAAA420
ATTG::TGATC
CCTCTCTCTA TAGGCGTTGT CTATCAAGGA AGATGGCTGA AGAACAACGT480
TGTTCTTCTG
AGTGGAATTA TGACTTACGC CCTTGGCAAG AAGAAGTGAT GCATTTGTTA540
GAGGAAGAAC
CAGATTATAG AACGATAATC TGGGTGTATG GACCTGCTGG TAATGAAGGC600
AAATCTACAT
TTGCAAGACA TCTGTCATTG AAAGATGGTT GGGGTTATCT GCCTGGAGGA660
AAGACACAAG
ATATGATGCA TCTTGTGACT GCTGAGCCTA AGAATAATTG GGTATTTGAC720
ATACCCAGAG
TTAGTTCAGA GTATGTGAAT TATGGTGTAA TAGAACAGGT TAAGAATAGG780
GTAATGGTGA
ATACTAAGTA TGAGCCATGT GTAATGCGGG ATGATAATCA TCCTGTTCAT840
GTAATTGTGT
TTGCAAATGT ACTCCCAGAT TTGGGAAAAT TAAGTGAAGA TAGAATAAAA900
TTAATTCGTT
GTTGAAAACT CTGCGAAGGC AGAAGTTATA AAAAAAATGT GTTTTGAGAG960
AAGTCCCACA
TCGGGTAGTT CGCGAAACAG GGTGAGGGAA GCGAGCAATA TAAGGCGAGG1017
TGCGTAT
SUBSTITUTE SHEET (RULE 26)
WO 96/06932 '~ ~ PCTlAU95/00552
-63-
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 988 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
TAGTATTACC CCGTGCCGGG ATCAGAGACA TTTGACTAAA TATTGACTTG GAATAATAGC 60
CCTTGGATTA GATGACACGT GGACGCTCAG GATCTGTGAT GCTAGTGAAG CGCTTAAGCT 120
GAACGAATCT GACGGAAGAG CGGACATACG CACATGGATT ATGGCCCACA TGTCTAAAGT 180
GTATCTCTTT ACAGCTATAT TGATGTGACG TAAGATGCTT TACTTCGCTT CGAAGTAAAG 240
TAGGAAATTG CTCGCTAAGT TATTCTTTTC TGAAAGAAAT TAATTTAATT CTAATTAAAT 300
TAAATGAGTG GCTATAAATA GTGTCGATGC TGCCTCACAT CGTATTCTTC TTCGCATCGT 360
CTGTTCTGGT TTTAAGCGAT GGTCAGTTTT AGTTTTCCTG AGATATACGA TGTGAGCGAC 420
GATGTTCTTG TAAGCGATAG CAGAAGAAGT GTAGCTGTTG AGGTCGAAGA GAAGGTTCAA 480
GTGATTAACG TGAAGGTACT GAGGTTGATT GAAGCTGTTG ATGAAGATAG AGTTGGAGTG 540
AAGGTTATGT TTCGTCTGTG TTACAGATAC AGACGAGAAC TGAAGATTAC GTTGTTGGGT 600
TGTAAGATGG AGCTATGGAC TTCGTTGAAG TCTTCAGGCA AGTATTCAGT TCAATCTTTG 660
TTGCAGAGGA AGCTTAATGG TATATGTGTT AGTAATTACT GTATAGGTAT TGATATGTTT 720
GTAAGTAATG TTAAAGAGTT GATTAATAGA TGTAAATGGA TTACATCTGT TCAAGGTGTT 780
AATCCTATAT GTTGTTTGTA TCATATGGAC GAAGAGTAAT TAATAGTAAT TATGATTAAT 840
TATGAGATAA GAGTTGTTAT TAATGCTTAT GAGGAATAAA GAATGATTAA TATTGTTTAA 900
TTTTATTCGC GAAGCGGTGT GTTATGTTTT TGTTGGAGAC ATCACGTGAC TCTCACGTGA 960
TGTCTCCGCG ACAGGCTGGC ACGGGGCT 9gg
SUBSTITUTE SHEET (RULE 26)
