Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1997-06-09
2001-04-03
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S320100, C435S468000, C536S024100, C800S280000, C800S288000, C800S298000, C800S301000, C800S302000
Reexamination Certificate
active
06211431
ABSTRACT:
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 nanovirus 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 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.
BRIEF DESCRIPTION OF THE INVENTION
Accordingly, one aspect of the present invention is directed to a genetic construct comprising a nanovirus promoter or promoter-like sequence and which is operable in a plant cell wherein said nanovirus comprises a multicomponent DNA genome.
In a related aspect of the present invention, there is provided a genetic construct comprising a nanovirus promoter or promoter-like sequence and a termination and/or polyadenylation sequence, which sequences are operable in a plant cell.
A nanovirus 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 nanovirus group is considered to include subterranean clover stunt virus (SCSV) (Chu and Helms, 1988), banana bunchy top virus (BBTV) (Thomas and Dietzgen, 1991; Harding etal. 1991; 1993; Bums etal. 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 nanovirus 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 subterranean clover stunt virus (hereinafter abbreviated to “SCSV”) as a representative of the nanovirus group. This is done, however, with the understanding that reference to SCSV includes reference to all other suitable members of the nanovirus group to which the instant invention extends. Preferred members of the nanovirus 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 romoter 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 nanovirus promoter or may contain one or more promoters in association with regulatory and/or reporter sequences.
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 ribozmes. 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 SF8;
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 r
Boevink Petra Christina
Chu Paul Wing Gay
Keese Paul Konrad
Khan Rafiqul Islam
Larkin Philip John
Commonwealth Scientific and Industrial Research Organization
McElwain Elizabeth F.
Mehta Ashwin D.
Scully Scott Murphy & Presser
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