Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-06-01
2001-10-02
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C536S023100
Reexamination Certificate
active
06297054
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of plant molecular biology. Specifically, DNA constructs are provided that facilitate the selection of stably transformed plastids in multicellular plants for which the encoded RNA is modified post-transcriptionally.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application by author names and year of publication in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Genetic engineering of plants involves the development and application of technology for genetic transformation through the direct manipulation of the plant genome and plant gene expression by the introduction of novel DNA. One method of transformation employs a derivative of the tumor inducing (Ti) plasmid from the bacterium,
Agrobacterium tumefaciens.
Other methods utilize direct gene transfer into protoplasts using biolistics, electroporation, polyethylene glycol treatment.
While the incorporation of transforming DNA in the nucleus of plant cells is well known to those skilled in the art, transformation protocols that selectively identify transformed plastid DNA, to the exclusion of other genetic compartments have not yet been described. With the above-described methods, if plastid transformation only is desired, nuclear transformants may express the gene encoding the selectable marker and result in the generation of false positives.
The need for plastid-specific marker genes is based on this observation. In earlier work, selection for kanamycin resistance of pTNH32-bombarded tobacco leaves yielded a large number of nuclear transformants (Carrer et al., 1993). Indeed, recovery of nuclear gene transformants with other plastid kan genes (Cornelissen and Vandewiele 1989), and with promoterless kan constructs (Koncz et al. 1989) confirms that kanamycin resistant clones may be readily obtained by transformation with constructs that were not designed for expression in the nucleus. Additionally, nuclear gene transformants in tobacco may also be recovered by selection for spectinomycin resistance genes designed for expression in plastids.
Given the large number of plastid genomes in plant cells, the ability to select for the transformed genome in culture is a key element in achieving successful transformation. Selection markers have been identified by screening cultured plant cells for mutants resistant to various substances, such as antibiotics and herbicides. Such antibiotics and herbicides are listed in Table I, below. However, to date, a method has not been developed that will facilitate plastid transformation with the concomitant exclusion of the selection of nuclear transformants. The development of such a system minimizes the false positives that result when a nuclear transformation event occurs.
RNA editing is a process that post-transcriptionally alters RNA sequences. Until recently, it was believed that chloroplasts, in contrast to mitochondria, did not utilize RNA editing and that the prediction of amino acid sequences from the corresponding gene sequences was generally correct. While most chloroplast genes begin with the canonical ATG start codon, genes have been identified that encode an ACG at a position that corresponds to the 5′ terminal ATG in homologous genes in other species. Recently it has been shown that this ACG codon is not conserved at the mRNA level. It is converted to a functional AUG codon by C to U editing (Hoch et al., 1991). Most of the edited codons found to date, restore amino acids that are conserved in the corresponding peptides from chloroplasts of other species. This editing process is plastid specific. Genes edited in the plastid are not edited in the nucleus or other organelles of the plant. The present invention provides DNA constructs and methods to facilitate the selection of stably transformed plastids, based upon a requirement for RNA editing in the transforming constructs which occurs exclusively in the plastid. Targeted manipulation of the plastid genome can now be performed with greater ease. Such manipulations include gene replacement, gene deletion, insertion of foreign genes and expression of recombinant proteins in plastids.
SUMMARY OF THE INVENTION
This invention provides DNA constructs and methods for the selection of stably transformed plastids of multicellular plants. The DNA constructs of the invention can be used for the exclusive selection of plastid transformants. Nuclear transformants will not be selected with the constructs of the instant invention.
According to one aspect of the invention, chimeric DNA constructs are described containing an edited gene segment translationally fused to a selectable marker gene. Following editing at the RNA level, which occurs in the plastid, the selectable marker gene is expressed. Cells or tissues are maintained on the selection medium until they have reached a homoplasmic condition, in which substantially all of the plastids of the cell or tissue have been transformed.
In a preferred embodiment of the invention, the above described chimeric construct is incorporated into a vector containing the necessary homologous sequences for targeted integration into the plastid genome. The targeting segment is of sufficient size to promote homologous recombination with a pre-determined plastid genome sequence, thereby replacing that sequence in the genome of the transformed plastid. The vector may further comprise a foreign gene of interest to beneficially augment the phenotype of the plant. In yet another embodiment of the invention, the chimeric DNA constructs may contain sequences that direct tissue specific regulation of the foreign gene of interest.
The method of the present invention is generally applicable to the selection of stably transformed plastids in both monocotyledonous and dicotyledonous plants. Following selection, the cells or tissues expressing the selectable phenotype are regenerated into multicellular plants.
REFERENCES:
patent: 5451513 (1995-09-01), Maliga et al.
patent: 5877402 (1999-03-01), Maliga et al.
Elizabeth H. Harris, et al. Antibiotic Resistance Mutations in the Chloroplast 16S and 23S rRNA Genes of Chlamydomonas reinhardtii: Correlation of Genetic and Physical Maps of the Chloroplast Genome. Genetics 123: 281-292 (Oct. 1989).
Sumita Chaudhuri, et al. Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids. The EMBO Journal. vol. 14, No. 12: 2951-2957 (1995).
Hillel Fromm, et al. The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts. The EMBO Journal vol. 6, No. 11: 3233-3237 (1987).
Wilhelm Gruissem. Chloroplast Gene Expression: How Plants Turn Their Plastids On. Cell 56:161-170 (Jan. 27, 1989).
Pal Maliga, et al. Plastid engineering in land plants: a conservative genome is open to change. Phil. Trans. R. Soc. Lond. B 342: 203-208 (1993).
J.-D.Rochaix. Post-Transcriptional Steps in the Expression of Chloroplast Genes. Annu. Rev. Cell Biol. 8:1-28 (1992).
Kurt Weising, et al. Foreign Genes in Plants: Transfer, Structure, Expression, and Applications. Annu. Rev. Genet. 22: 421-77 (1988).
Hans Kossel, et al. RNA Editing in Chloroplasts of Higher Plants. Plant Mitochondria (Brennicke, A, and Kuck, U. eds.) VCH Verlagsgesellschaft mbH, Weinheim (Germany) (1993).
Pal Maliga. Towards Plastid Transformation in Flowering Plants. Tibtech vol. 11 (Mar. 1993).
Ralph Bock, et al. Introduction of a heterologous editing site into the tobacco plastid genome: the lack of RNA editing leads to a mutant phenotype. The EMBO Journal. vol. 13, No. 19: 4623-4628 (1994).
Ralph Bock, et al. In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL eidting. Mol. Gen Genet 247:439-443 (1995).
Zora Svab, et al. High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA 90:913-917 (Feb. 1993).
Cha
Carrer Helaine
Chaudhuri Sumita
Maliga Pal
Dann Dorfman Herrell and Skillman
Davis Katharine F
Rutgers The State University of New Jersey
Schwartzman Robert A.
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