Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide comprises a transposon
Reexamination Certificate
2000-06-01
2002-04-09
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide comprises a transposon
C800S266000, C800S270000, C800S298000, C435S419000, C435S468000
Reexamination Certificate
active
06369297
ABSTRACT:
BACKGROUND OF THE INVENTION
Transposable elements are powerful genetic tools for the cloning of genes on the basis of their phenotypes (Bingham et al., Cell 25:693-704, 1981). Introduction of the maize transposon Activator (Ac) into heterologous plant species has resulted in the isolation of various genes by transposon tagging. See, e.g., Aarts et al., Nature 363:715-717, 1993; Jones et al., Science 266:789-793, 1994; and Whitham et al., Cell 78:1101-1115, 1994. The use of an autonomous Ac element for gene tagging allows one to take advantage of the higher transposition activity of Ac associated with the homozygous state, and thereby to increase the possibility of insertion into a target gene.
SUMMARY OF THE INVENTION
The invention relates to the discovery of a transposition system requiring only a single inducible genetic component and that can be used for plant gene tagging in germinal tissue. The genetic component generally contains a promoter separated from an open reading frame (ORF) by an inducible transposable element. Transposition is then monitored by determining whether the protein encoded by the open reading frame is present in the plant or cell thereof.
Accordingly, the invention features a nucleic acid containing (1) an inducible transposable element including a first nucleotide sequence encoding a transposase (e.g., the transposase of a maize transposable element Activator) and an inducible promoter (e.g., a pathogenesis-related protein 1a promoter [PR-1a promoter; see Grüner et al., Eur. J. Biochem. 220:247-255, 1994]) operably linked to the first nucleotide sequence; (2) an uncoupled promoter (e.g., a 1′ promoter of a T-DNA 1′ gene); and (3) a second nucleotide sequence encoding a polypeptide (e.g., a detectable protein, such as luciferase). Upon removal of the inducible transposable element during transposition, the uncoupled promoter becomes operably linked to the second nucleotide sequence. The inducible transposable element can further include a selectable marker gene (e.g., one that expresses a hygromycin phosphotransferase).
The invention also includes a transgenic plant containing a nucleic acid of the invention, and a method of producing a progeny plant containing a transposition by (1) contacting a germinal tissue of a transgenic plant of the invention with an inducer (e.g., a chemical inducer) of the inducible promoter, (2) pollinating the germinal tissue, and (3) germinating seedlings from the transgenic plant to produce the progeny plant. A “germinal tissue” is any tissue that can give rise to an entire progeny plant, such as a flower tissue (e.g., the pollen, pollen mother, egg, or egg mother cells of the flower) or precursor tissues thereof. If the inducible promoter is a PR-1a promoter, then the inducer can be an aqueous solution containing at least about 0.5 mM (e.g., about 1 to 10 mM, about 5 mM, at least about 30 mM, and about 50 mM) salicylic acid.
By one genetic element being “operably linked” to another is meant that a genetic element (either in a plus strand, minus strand, or double stranded form) is structurally configured to operate or affect another genetic element. For example, a promoter operably linked to a sequence encoding a polypeptide means that the promoter initiates transcription of a nucleic acid encoding the polypeptide. An “uncoupled promoter” means that a promoter is not operably linked to a particular genetic element. A “selectable marker gene” is a gene that expresses a nucleic acid or protein which confers a readily observable (by any means known in the art) phenotype in a cell or plant harboring the marker gene. A “detectable protein” is a protein that is readily observable using any known means in the art of molecular biology, such as by fluorescence, phosphorescence, luminescence, radioactivity, enzymatic activity, or chromogenic activity.
The nucleic acids, transgenic plants, and methods described above are useful for generating mutated transgenic plants by inducing transposon insertion in germinal tissue. These mutants can then be screened for particular phenotypes. An examination of the site of insertion in these mutants can help identify a gene that is involved in the particular phenotype.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.
DETAILED DESCRIPTION
The invention is based on a single nucleic acid construct which, after integration into a plant genome using standard methods, can be used to induce a transposition from one position in a genome to another in a germ line cell. The transposition is observable upon removal of the transposon from its site in the construct, the removal bringing together a previously uncoupled promoter and an ORF that expresses, e.g., a detectable protein.
REFERENCES:
patent: 5013658 (1991-05-01), Dooner et al.
Charng, et al, “ A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase . . . ”, 1997, Plant Science, vol. 130 pp. 73-86.*
Charng, et al, “Fusion of the inducible promoter of the PR-1a gene to the Activator transposase gene can transactive excision of non-atonomous . . . ”, 1995, Plant Science vol. 106 pp. 141-155.*
Jones et al., “Visual Detection . . . ,” Science, 244:204-207, 1989.
Finnegan et al., “Transposable Elements . . . ,” The Plant Cell, 1:757-764, 1989.
Baker et al., “Phenotypic assay . . . ,” The EMBO Journal, 6:1547-1554, 1987.
Charng et al., “The firefly luciferase . . . ,” Plant Science, 98:175-183, 1994.
Charng et al., “Fusion of the inducible . . . ,” Plant Science, 106:141-155, 1995.
Academia Sinica
Collins Cynthia
Fish & Richardson P.C.
McElwain Elizabeth F.
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