Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-08-14
2004-08-24
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S288000, C800S294000, C800S306000, C435S008000, C435S069700, C435S430000, C435S430100, C435S463000, C435S468000, C435S469000
Reexamination Certificate
active
06781032
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to methods for transforming plant cells and, more particularly, to methods for identifying plants in which a target DNA sequence has been modified by homologous recombination.
BACKGROUND OF THE INVENTION
Efficient methods for identifying plant cells in which homologous recombination has occurred is extremely useful in developing new plant varieties with targeted replacement or alteration of endogenous genes. Activation of a reporter gene activity due to homologous recombination between two non-allelic mutant copies of a reporter gene or between two overlapping deletions of a reporter gene have been reported in the literature (see, Assaad, et al.,
Genetics
, 132:553-566 (1992); Tovar, et al.,
Plant Cell
, 4:319-332 (1992); Baur, et al.,
Mol. Cell. Biol
., 10:492-500 (1990); Chiurazzi, et al.,
Plant Cell
, 8(11):2057-2066 (1996); Hrouda, et al., Mol. Gen. Genet. 243:106-111 (1994); Lebel, et al.,
Proc. Natl. Acad. Sci. USA
, 90:422-426 (1993); Lyznik, et al.,
Mol. Gen. Genet
. 230:209-218 (1991); Offringa, et al.
Proc. Natl. Acad. Sci. USA
, 90:7346-7350 (1993); Offringa, et al.,
EMBO J
, 9:3077-3084 (1990); Swoboda, et al.,
EMBO J
, 13:484-489 (1994); Risseeuw, et al.,
Plant J
, 7:109-119 (1995); Puchta, et al.,
Proc. Natl. Acad. Sci. USA
93:5055-5060 (1996); Puchta, et al.,
Plant Mol. Biol
. 28:281-292 (1995); Puchta, et al.,
Nuc. Acids Res
., 21:5034-5040 (1993); Puchta, et al.,
Mol. Cell. Biol
., 12:3372-3379 (1992); Puchta, et al.,
Nuc. Acids Res
. 19:2693-2700 (1991); Puchta, et al.,
Mol. Gen. Genet
., 230:1-7 (1991); Paszkowski, et al.,
EMBO J
, 7:4021-4026 (1988); Peterhans, et al.,
EMBO J
., 9:3437-3446 (1990)). However, these reports are limited to identifying recombination events within genes which themselves had some inherent “reporter gene activity” (e.g. drug resistance or conversion of a calorimetric substrate). In most cases, the homologous recombination events were non-reciprocal (unidirectional) homology dependent events, referred to as gene conversion events.
Several reports in animal gene targeting experiments use a dominant selectable reporter gene as part of a &Dgr;gene X::reporter gene fusion to select for homologous recombination events in vivo (Buerstedde, et al.,
Cell
, 67:179-88 (1991); Jasin, et al.,
Genes and Devel
., 2:1353-63 (1988); Sedivy, et al.,
Proc. Natl. Acad. Sci. USA
, 86:227-31 (1989)). In all cases, the reporter genes used imparted resistance to a drug that was used as a dominant positive selection for those cells that underwent the specific homologous recombination between the &Dgr;gene X::reporter gene and the endogenous gene. A dominant selection approach can be problematic because either an insufficient level of the drug resistance trait or a tissue specific gene X expression pattern of the drug resistance trait may result in an inability to survive the drug selection during cell culture. The same concerns regarding the level and tissue specific expression pattern of the recombinant gene X′/X::reporter gene apply to the situations of using a dominant selectable reporter gene on a whole organism-based screen.
Although progress has been made, efficient screening methods for detecting homologous recombinants, particularly in plant cells is needed in the art. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods which use a reporter sequence to identify homologous recombinants in a large population of transformed plants or plant cells. The methods of the invention involve contacting plant cells with a nucleic acid molecule comprising a fusion polynucleotide sequence comprising a polypeptide sequence of interest linked to a reporter sequence, wherein the nucleic acid molecule lacks sequences necessary for expression of the fusion polynucleotide sequence gene product in a cell. The fusion polynucleotide sequence gene product is then detected in the plant cells, thereby identifying plant cells in which homologous recombination has occurred.
The means by which the fusion polynucleotide is introduced into the cell is not critical. Typically, the polynucleotide is introduced using a T-DNA vector. In some embodiments, plants are regenerated from the plant cells before the step of detecting the presence of the fusion sequence gene product.
The particular reporter sequence used in the methods is also not critical, for instance, non-selective markers such as luciferase can be used. In this case video imaging equipment is conveniently used to detect the luciferase.
The homologous recombination event can be used to alter endogenous gene in any number of ways. For instance, the recombination can result in gene conversion or may lead to inactivation of the endogenous gene. Alternatively, a recombinant allele derived from two related genes can be produced. The newly created recombinant allele will typically have a new activity as compared to either of the genes from which it was derived.
The invention also provides isolated nucleic acid molecule useful in the above methods, as well as plants produced by the methods.
Definitions
The term “homologous recombination” refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity. The term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion). In addition, the recombination can be the result of equivalent or non-equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions. For a description of the enzymes and mechanisms involved in homologous recombination see, Watson et al.,
Molecular Biology of the Gene
pp 313-327, The Benjamin/Cummings Publishing Co. 4th ed. (1987).
The phrase “nucleic acid sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. As used herein, a “plant promoter” is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term “plant” includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells and their progeny. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign speci
Furuya Masaki
Gruissem Wilhelm
Jelesko John
Fox David T.
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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