Maize DNA ligase I orthologue and uses thereof

Details Maize DNA ligase I orthologue and uses thereof Maize DNA ligase I orthologue and uses thereof

1999-10-22

2001-02-27

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S286000, C800S306000, C800S314000, C800S312000, C800S320000, C800S320100, C800S320300, C800S322000, C800S320200, C435S069100, C435S069200, C435S468000, C435S419000, C435S430000, C435S431000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024500

Reexamination Certificate

active

06194637

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
DNA ligase is an important component of the machinery that all living cells use to maintain the integrity of their genetic material. This enzyme catalyzes the ligation of DNA strand breaks generated during various cellular metabolic events such as DNA replication and recombination. It also plays a crucial role in repairing DNA strand breaks generated in response to various environmental insults (1, 2). Higher eukaryotes have more than one type of DNA ligase. In fact, five distinct ligase activities have been reported in humans. Three human ligase genes LIG I, LIG II and LIG III have also been characterized (3). Ligase II is believed to be a proteolytic product of Ligase I. Ligase IIIa and IIIb are produced from LIG III by differential processing. In addition to being encoded by different genes, these ligases also exhibit differential substrate specificity and are localized in different cellular compartments. Therefore, it has been proposed that they are involved in different metabolic functions in eukaryotic organisms (1-4).
During the past three decades, DNA ligase genes have been cloned from a large number of organisms (1-4). The enzyme from higher eukaryotes is very similar to that from the viral or phage enzyme in its requirement for ATP. Bacterial enzymes require NAD as a co-factor. Nonetheless, all ligases share many common structural motifs characteristic of this family of proteins. Despite these similarities, there are important differences in the primary structure of DNA ligases from different species. This may account for the difficulty of cloning by homology of plant DNA ligases. The only plant DNA ligase cloned as of 1998 is from
Arabidopsis thaliana
. The maize DNA Ligase I of the present invention is the first ligase to be cloned from maize.
The need for the detailed characterization of maize DNA ligases for use in DNA integration is clear. Further, what is needed in the art is a means to improve transformation efficiency. The present invention provides 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 DNA ligase I. 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, wherein the polypeptide when presented as an immunogen elicits the production of an antibody which is specifically reactive to the polypeptide; (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 as described, supra, operably linked to a promoter. In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette as described, supra. In some embodiments, the host cell is a sorghum (
Sorghum bicolor
) or maize (
Zea mays
) cell.
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 the isolated nucleic acid referred to, supra.
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 nucleic acid of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In yet another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide, the polynucleotide having a specified sequence identity to an identical length of a nucleic acid of the present invention or a complement thereof.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide having a sequence of a nucleic acid amplified from a
Zea mays
nucleic acid library using at least two primers or their complements, one of which selectively hybridizes under stringent conditions to a locus of the nucleic acid comprising the 5′ terminal coding region and the other primer selectively hybridizing, under stringent conditions, to a locus of the nucleic acid comprising the 3′ terminal coding region, and wherein both primers selectively hybridize within the coding region. In some embodiments, the nucleic acid library is a cDNA library.
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 an additional aspect, the present invention is directed to an isolated nucleic acid comprising a polynucleotide encoding a polypeptide wherein: (a) the polypeptide comprises a specified number of contiguous amino acid residues from a first polypeptide of the present invention, wherein the polypeptide, when presented as an immunogen, elicits the production of an antibody which specifically binds to said first polypeptide; (b) the polypeptide does not bind to antisera raised against the first polypeptide which has been fully immunosorbed with the first polypeptide; (c) the polypeptide has a molecular weight in non-glycosylated form within a specified percentage of the first polypeptide.
In a further aspect, the present invention relates to a heterologous promoter operably linked to a non-isolated polynucleotide of the present invention, wherein the polypeptide is encoded by a nucleic acid amplified from a nucleic acid library.
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. In some embodiments, the transgenic plant is
Zea mays
. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of modulating expression of the genes encoding the proteins of the present invention in a plant, comprising the steps of (a) transforming a plant cell with a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; (b) growing the plant cell under plant growing conditions; and (c) inducing expression of the polynucleotide for a time sufficient to modulate expression of the genes in the plant. In some embodiments, the plant is maize. Expression of the genes encoding the proteins of the present invention can be increased or decreased relative to a non-transformed control 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 rang

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