Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
2000-02-16
2002-05-21
LeGuyader, John L. (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C435S410000, C435S320100, C536S023200
Reexamination Certificate
active
06392126
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. In particular, the present invention relates to nucleic acids encoding polypeptides which function as adenosine deaminases.
BACKGROUND OF THE INVENTION
Adenosine deaminase (abbreviated hereafter as ADA; EC 3.5.4.4.) catalyzes the irreversible deamination of adenosine and 2′deoxyadenosine to the corresponding inosine derivatives. This enzyme is indispensable for purine metabolism. ADA deficiency results in severe combined immunodeficiency disease or SCID, a genetic disorder. Substrate accumulation leading to an interference with DNA synthesis underlies the damage occurring in ADA deficiency (Giblett, E.
in Annals of the New York Academy of Sciences,
NY Acad. Sci., New York 451: 1-8, 1985). Levels of ADA are also affected in a variety of leukemias, lymphomas, AIDS, and anemia (Adenosine Deaminase in Disorders of Purine Metabolism and in Immune Deficiency, ed. George L. Tritsch,
Annals of the New York Academy of Sciences
Vol. 451, New York Acad. of Sci., New York, N.Y., 1985). Consequently, ADA has been studied in detail in mammalian systems (Daddona, P E and Kelley, W N
Mol. Cell. Biochem.
29: 91-101, 1980; Wilson, D K et al.
Science
252: 1278-1284, 1991; Hirschhorn, R.
Pediatric Res.
33 (Supplement): 35-41, 1993; Markert, M L
Immunodeficiency
5, 141-157, 1994; Morgan R A and Anderson, W F
Ann Rev. Biochem.
62: 191-217, 1993; Vega, M A
Biochim. Biophys. Acta
1138: 253-260, 1992). ADA has also been studied in invertebrates, bacteria and fungi (Schomburg, D. and Salzmann, M.
in The Enzyme Handbook Vol
4 Springer Verlag, Berlin, 1991). ADA activity has not been detected, however, in plants such as barley, cucumber, artichoke, lupin or in seeds from 17 different plant species. It has even been stated that plants do not contain ADA (Dancer J E et al.
Plant Physiol.
114: 119-129, 1997). Therefore, it is generally accepted that the ADA gene is absent in multicellular plants or is expressed only under tight developmental control (Pelcher, L. U.S. Pat. No. 5,474,929, 1993). Tight control of ADA expression has been shown in mouse tissue where the expression of ADA has been found to be governed in both a cell-specific and developmentally regulated manner (Chinsky J M et al.
Differentiation
42: 172-183, 1990).
The maize ADA gene of the present invention is the first ADA gene reported to exist in a multicellular plant. The existence of maize ADA provides for many advantages. Maize ADA would be useful as a selectable marker in transformation experiments. Most of the genes which are currently available for use in the selection of transformed plants or plant cells are either antibiotic- or herbicide-resistance genes. There is a need for more selectable marker genes. The growth of tobacco leaf explants has been shown to be inhibited by 2′-deoxyadenosine and analogs of deoxyadenosine (Pelcher, L. U.S. Pat. No. 5,474,929, 1993). Tobacco cells transformed with an ADA gene from a mouse cell line have been shown to proliferate on media containing an analog of deoxyadenosine (Pelcher, L. U.S. Pat. No. 5,474,929, 1993). Thus, the ADA gene has been shown to be useful in the selection of transformed plant cells.
The maize ADA cDNA sequences provided in the present invention include 3′-untranslated regions. These regions are suitable for use in making gene-specific probes which can be used to map the locus of a maize ADA gene in an RFLP map of a maize population. The present invention provides for the use of the ADA gene as a selectable marker, the use of the 3′-untranslated regions as gene-specific probes, along with other advantages.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to maize ADA. It is an object of the present invention to provide: 1) antigenic fragments of the proteins of the present invention; 2) transgenic plants comprising the nucleic acids of the present invention; 3) methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and, (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having a specified number of contiguous amino acids encoded by an isolated nucleic acid of the present invention.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of specified length which selectively hybridizes under stringent conditions to a polynucleotide of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid amplified from a library as referred to supra, wherein the nucleic acid is operably linked to a promoter. In some embodiments, the present invention relates to a host cell transfected with this recombinant expression cassette. In some embodiments, the present invention relates to a protein of the present invention which is produced from this host cell.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. The present invention also provides transgenic seed from the transgenic plant.
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 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. 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.
The term “antibody” includes reference to antigen binding forms of antibodies (e.g., Fab, F(ab)
2
). The term “antibody” frequently refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof whi
LeGuyader John L.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
Schmidt M
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