Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-03-25
2002-04-30
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S278000, C800S295000, C800S298000, C800S286000, C435S069100, C435S320100, C435S419000, C435S468000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024500
Reexamination Certificate
active
06380459
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to the regulation of gene transcription and/or expression. More specifically, this invention relates to polynucleotide regulatory sequences isolated from plants that are capable of initiating and driving the transcription of genes, and the use of such regulatory sequences in the modification of transcription of endogenous and/or heterologous genes.
BACKGROUND OF THE INVENTION
Gene expression is regulated, in part, by the cellular processes involved in transcription. During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polymerases. Initiation of transcription in eucaryotic cells is regulated by complex interactions between cis-acting DNA motifs, located within the gene to be transcribed, and trans-acting protein factors. Among the cis-acting regulatory regions are sequences of DNA, termed promoters to which RNA polymerase is first bound, either directly or indirectly As used herein, the term “promoter” refers to the 5′ untranslated region of a gene that is associated with transcription and which generally includes a transcription start site. Other cis-acting DNA motifs, such as enhancers, may be situated further up- and/or down-stream from the initiation site.
Both promoters and enhancers are generally composed of several discrete, often redundant, elements each of which may be recognized by one or more trans-acting regulatory proteins, known as transcription factors. Promoters generally comprise both proximal and more distant elements. For example, the so-called TATA box, which is important for the binding of regulatory proteins, is generally found about 25 basepairs upstream from the initiation site. The so-called CAAT box is generally found about 75 basepairs upstream of the initiation site. Promoters generally contain between about 100 and 1000 nucleotides, although longer promoter sequences are possible.
For the development of transgenic plants, constitutive promoters that drive strong transgene expression are preferred. Currently, the only available constitutive plant promoter that is widely used is derived from Cauliflower Mosaic Virus. Furthermore, there exists a need for plant-derived promoters for use in transgenic food plants due to public conceptions regarding the use of viral promoters. Few gymnosperm promoters have been cloned and those derived from angiosperms have been found to function poorly in gymnosperms. There thus remains a need in the art for polynucleotide promoter regions isolated from plants for use in modulating transcription and expression of genes in transgenic plants.
SUMMARY OF THE INVENTION
Briefly, isolated polynucleotide regulatory sequences from eucalyptus and pine that are involved in the regulation of gene expression are disclosed, together with methods for the use of such polynucleotide regulatory regions in the modification of expression of endogenous and/or heterologous genes in transgenic plants. In particular, the present invention provides polynucleotide promoter sequences from 5′ untranslated regions of plant genes that initiate and regulate transcription of DNA sequences placed under their control.
In a first aspect, isolated polynucleotide promoter sequences are provided that comprise a DNA sequence selected from the group consisting of: (a) sequences recited in SEQ ID NO: 2-14 and 20; (b) complements of the sequences recited in SEQ ID NO: 2-14 and 20; (c) reverse complements of the sequences recited in SEQ ID NO: 2-14,20; (d) reverse sequences of the sequences recited in SEQ ID NO: 2-14 and 20; and (e) sequences having either 40%, 60%, 75% or 90% identical nucleotides, as defined herein, to a sequence of (a)-(d).
In a related aspect, the present invention provides DNA constructs comprising, in the 5′-3′ direction, a polynucleotide promoter sequence of the present invention, a DNA sequence to be transcribed, and a gene termination sequence. The DNA sequence to be transcribed may comprise an open reading frame of a DNA sequence that encodes a polypeptide of interest or may be a non-coding, or untranslated, region of a DNA sequence of interest. The open reading frame may be orientated in either a sense or antisense direction. Preferably, the gene termination sequence is functional in a host plant. Most preferably, the gene termination sequence is that of the gene of interest, but others generally used in the art, such as the
Agrobacterium tumefaciens
nopalin synthase terminator may be usefully employed in the present invention. The DNA construct may further include a marker for the identification of transformed cells.
In a further aspect, transgenic plant cells comprising the DNA constructs of the present invention are provided, together with organisms, such as plants, comprising such transgenic cells, and fruits and seeds of such plants.
In yet another aspect, methods for modifying gene expression in a target organism, such as a plant, are provided, such methods including stably incorporating into the genome of the organism a DNA construct of the present invention. In a preferred embodiment, the target organism is a plant, more preferably a woody plant, most preferably selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of
Eucalyptus grandis
and
Pinus radiata.
In another aspect, methods for producing a target organism, such as a plant, having modified gene expression are provided, such methods comprising transforming a plant cell with a DNA construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.
In other aspects, methods for identifying a gene responsible for a desired function or phenotype are provided, the methods comprising transforming a plant cell with a DNA construct comprising a polynucleotide promoter sequence of the present invention operably linked to a gene to be tested, cultivating the plant cell under conditions conducive to regeneration and mature plant growth to provide transgenic a plant; and comparing the phenotype of the transgenic plant with the phenotype of non-transformed, or wild-type, plants.
In yet a further aspect, the present invention provides an isolated polynucleotide from
Pinus radiata
that encodes ubiquitin. In specific embodiments, the isolated polynucleotide comprises a DNA sequence selected from the group consisting of: (a) a sequence recited in SEQ ID NO: 1; (b) complements of the sequence recited in SEQ ID NO: 1; (c) reverse complements of the sequence recited in SEQ ID NO: 1; (d) reverse sequences of the sequence recited in SEQ ID NO: 1; and (e) sequences having either 40%, 60%, 75% or 90% identical nucleotides, as defined herein, to a sequence of (a)-(d). Polypeptides encoded by such polynucleotides are also provided, together with DNA constructs comprising such polynucleotides, and host cells and transgenic organisms, for example plants, transformed with such DNA constructs.
In yet further aspects, the present invention provides isolated polynucleotides comprising the DNA sequence of SEQ ID NO: 21, or a complement, reverse complement or variant of SEQ ID NO: 21, together with DNA constructs comprising such polynucleotides and cells transformed with such sequences. As discussed below, removal of the sequence of SEQ ID NO: 21 from a polynucleotide that comprises the sequence of SEQ ID NO: 21 may enhance expression of the polynucleotide. Conversely, the inclusion of the sequence of SEQ ID NO: 21 in a DNA construct comprising a polynucleotide of interest may decrease expression of the polynucleotide.
The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
DETAILED DESCRIPTION OF THE
Perera J. Ranjan
Rice Stephen J.
Bui Phuong T.
Genesis Research & Development Corporation Ltd.
Ibrahim Medina A.
Sleath Janet
Speckman Ann W.
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