Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2001-01-05
2003-12-02
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C536S023100, C536S024100, C536S024500, C435S320100, C435S419000, C435S069100, C435S091400, C800S295000
Reexamination Certificate
active
06657107
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
A variety of environmental agents such as gamma radiation, UV light in the 320-380 nm range, ozone, heat, and various chemicals cause oxidative damage to cellular DNA. Similarly, reactive oxygen species, hydroxyl radicals and superoxide and nitric oxide species generated in vivo cause oxidative damage to DNA (Friedberg, E. et al., in
DNA repair and Mutagenesis, American Society of Microbiology Press
, Washington D.C., pages 14-19, 1995). The precise nature of DNA modification varies depending upon the exposure and type of causative reagent. Such modifications as breakage of the phosphodiester bond have been reported, as well as oxidative stress induced illegitimate recombination in bacteria (Ouchane S. et al.,
EMBO J
. 16: 4777-4787,1997). However, the most common result of oxidative damage is the oxidation of bases and sugars. Formamidopyrimidine (Fapy), 8-hydroxyguanine and 8-oxo-7,8 dihydrodeoxyguanosine are the most commonly observed base modifications following oxidative damage. Of these, 8-hydroxyguanine is considered highly mutagenic. It causes G:C to A:T transversions because 8-hydroxyguanine can pair with adenine and cytosine nucleotides with almost equal efficiencies during DNA replication (Shibutani A. et al.,
Nature
349: 431434, 1991; Maki, H. and Sekiguchi M.,
Nature
355: 273-275,1992).
Consequently, all living organisms have developed specific enzymatic pathways to remove such lesions and to maintain genomic stability. These enzymatic pathways have been very well characterized in bacteria and lower eukaryotes such as yeast. Implications of the involvement of oxidative DNA damage in the development of malignancies have also prompted a detailed analysis of these pathways in mammalian systems such as humans. These pathways have not been well studied however, in plants such as maize.
In
E. coli
, three genes labeled mutM, mutY, and mutT encode the enzymes responsible for the removal of Fapy and 8-hydroxyguanine lesions. Their gene products are members of the DNA glycosylase family. The mutY gene product specifically removes the unmodified A from the 8-hydroxyguanosine: A pair. The mutT gene product, on the other hand, preferentially hydrolyzes 8-oxo-7,8 dihydrodeoxyguanosine thereby preventing its incorporation in DNA.
E. coli
mutants of these genes show a mutator phenotype with a 10-1000 fold increase in transversions compared to wild type. In addition to the mutator phenotype,
E.coli
mutM mutants show increased illegitimate or non-homologous recombination. Furthermore, the mutM gene product suppresses this illegitimate recombination (Onda, M. et al.,
Genetics
151:439446, 1999). Thus, overexpression of the mutM gene product may be used as a tool to suppress mutations in general and oxidative stress induced non-homologous recombination in particular.
Recent studies have revealed the presence of mutM orthologues in yeast, human, and
Arabidopsis thaliana
(van der Kemp PA et al.,
PNAS
93:5197-5202, 1996; Arai, K. et al.,
Oncogene
14:2857-2861,1997; Radicella, JP et al.,
PNAS
94:8010-8015,1997; Ohtsubo, T. et al.,
Mol. Gen. Genet
. 259:577-590,1998). The present invention presents a full-length cDNA encoding a maize orthologue of
E. coli
mutM. Unlike the animal mutM orthologues, the maize enzyme contains a C-terminal region of alternating acidic and basic amino acid residues and a putative nuclear localization signal as shown in Example 4. The mutM orthologue of the present invention may be useful as a suppresser of DNA mutations which are induced by oxidative damage. Furthermore, it may be used to reduce illegitimate recombination thereby increasing frequencies of homologous recombination and transformation. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides for these and other advantages.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to maize mutM. It is an object of the present invention to provide expression cassettes, host cells and transgenic plants comprising the nucleic acids of the present invention, and methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention in order to improve the efficiency of homologous recombination, transformation efficiency or to induce targeted gene changes. It is also an object of the present invention to provide antibody compositions for detecting the polypeptides of the present invention.
In other aspects the present invention relates to: 1) recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter, 2) a non-human host cell into which has been introduced the recombinant expression cassette, and 3) a transgenic plant comprising the recombinant expression cassette.
Definitions
Units, prefixes, and symbols may be denoted in their Si accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5
th
edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g.,
Diagnostic Molecular Microbiology: Principles and Applications
, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
As used herein, “antisense orientation” includes reference to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.
By “encoding” or “encoded”, with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as are present in some plant, animal, and fungal mitochondria, the bacterium
Mycoplasma capricolum
, or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.
When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host
Bui Phuong T.
Helmer Georgia
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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