Maize Rad2/FEN-1 orthologues and uses thereof

Details Maize Rad2/FEN-1 orthologues and uses thereof Maize Rad2/FEN-1 orthologues and uses thereof

1999-10-22

2001-05-15

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S286000, C800S320000, C800S320100, C800S320200, C800S306000, C800S314000, C800S322000, C800S295000, C800S298000, C800S320300, C800S312000, C435S069100, C435S320100, C435S419000, C435S468000, C536S023100, C536S023600, C536S024100, C536S024500

Reexamination Certificate

active

06232527

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
Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome such problems as expression variability, unpredictable impacts of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted to obtain ideal transgenic plants. Such systems can also provide approaches to manipulating endogenous genes.
Gene targeting systems require the ability to focus the recombination process to favor the recovery of desired targeting events. The natural cellular DNA repair and recombination machinery consists of a complex array of protein components interacting in a highly controlled manner to ensure that the fidelity of the genome is conserved throughout the many internal events or external stimuli experienced during each cell cycle. The ability to manipulate this machinery requires an understanding of how specific proteins are involved in the process, and how the genes that encode those proteins are regulated. Because many different protein components may be involved in gene targeting, the availability of host-specific genes and proteins could avoid possible problems of incompatibility associated with molecular interactions due to heterologous components.
The RAD2 gene of the budding yeast
Saccharomyces cerevisiae
is one of several genes known to be important in excision repair (1). It encodes an endonuclease that specifically cleaves single-stranded DNA in the 5′ to 3′ orientation. The yeast RAD2 gene and encoded protein (Rad2) exhibit high homology to a human DNA repair protein XP-G (2) and the structure specific Flap Endonuclease-1 or FEN-1 (3, 4). Human FEN-1 is a 380 amino acid protein which cleaves DNA flap strands that terminate with 5′ single strand ends. This cleavage is flap strand specific and independent of the flap strand sequence or length (3). Other branched structures such as Holliday junctions or double D loop are not cleaved by FEN-1 (3). Members of this family have also been cloned from the budding yeast (5), Xenopus (6), and mouse (7), as well as the archaebacteria (8). Recently, Kimura et al. have also characterized a structure-specific endonuclease from
Brassica oleracea
(9).
Genetic and biochemical studies have established that the Rad2/FEN-1 protein is a structure specific endonuclease (8, 10). Moreover, under certain reaction conditions, it also acts as an exonuclease (8, 10). The endonucleolytic activity is essential in DNA replication as well as the nucleotide excision repair reactions (8, 10). The exonucleolytic activity is involved in double strand break repair and end joining (8, 10). The protein is also useful in strand exchange reactions during homologous recombination (8, 10). These functions could prove to be very useful in gene targeting and in the production of male sterile plants. For example, the efficiency of gene targeting can be improved by the overexpression of exogenous Rad2/FEN-1 while male sterile plants can be produced by the down-regulation of Rad2/FEN-1 expression.
The regulation of DNA repair and recombination in plant systems by the modulation of maize Rad2/FEN-1 will provide improved and expanded methods of gene targeting. The need in the art for methods to regulate gene targeting and to modulate male sterility is clear. 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 Rad2/FEN-1. 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 which specifically bind and recognize an analyte (antigen). However, while various antibody fragments can be defined in terms of the digestion

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