Mre11 orthologue and uses thereof

Details Mre11 orthologue and uses thereof Mre11 orthologue and uses thereof

2001-04-16

2003-11-11

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S320100, C435S419000, C435S069100, C536S023100, C536S024100, C536S024500, C800S295000, C800S312000, C800S314000, C800S317000, C800S320000, C800S320100

Reexamination Certificate

active

06646182

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
In 1993, Ajimura et al. isolated a temperature sensitive mutant of
S. cerevisiae
that was shown to be defective in initiation of meiotic recombination. This mutant, mre11-1, was sensitive to the radiomimetic agent methyl methanesulfonate (MMS) and showed a 10-fold increase in the level of mitotic recombination (Ajimura M et al.,
Genetics
133: 51-66, 1993). Based on these properties, the MRE11 gene has been classified as belonging to same epistasis group as RAD50. A null mutant of MRE11 is unable to initiate meiosis, rendering the spores non-viable. The Mre11 protein has been shown to interact with the Rad50 protein to initiate double strand breaks in meiotic recombination (Johzuka K and Ogawa H.,
Genetics
139: 1521-1532, 1995). A new mutant allele, mre11S, was isolated and shown to block processing but not formation of double strand breaks (Nairz K and Klein F,
Genes and Dev.
11: 2272-2290, 1997). Another mutant Mre11 allele which has been characterized, mre11-58, has been shown to contain two amino acid changes from the wild type protein. Interestingly, unlike mre11 null mutants, mre11-58 was proficient in formation of double strand breaks, but defective in processing of the DNA ends, indicating the involvement of Mre11 protein in exonucleolytic processing of double strand breaks during meiosis (Tsubouchi H and Ogawa H,
Mol. Cell. Biol.
18: 260-268, 1998). This 3′ to 5′ exonuclease activity of Mre11 on double-stranded DNA either by itself, or in complex with Rad50 and Xrs2/p95 has been clearly established by two different groups (Paull T and Gellert M,
Mol. Cell
1: 969-979, 1998; Trujillo K M et al.,
J Biol Chem
272: 21447-21450, 1998). The exonuclease activity is observed only in the presence of Mn
++
. Mre11 also exhibits Mn
++
-dependent endonuclease activity on ssDNA (Trujillo K M et al.,
J Biol Chem
273: 21447-21450, 1998) as well as on hairpin loops formed during V(D)J recombination (Paull T and Gellert M,
Mol Cell
1: 969-979, 1998).
The involvement of the MRE11/RAD50/XRS2 group of genes in non-homologous end joining (also known as non-homologous or illegitimate recombination) has also been well documented (Moore J K and Haber J E,
Mol. Cell Biol.
16: 2164-2173, 1996; Tsukamoto Y et al.,
Genetics
142:383-391, 1996; Wilson S, et al.,
Nucleic Acid Res
27: 2655-2661, 1999; Lewis L K et al.,
Genetics
152: 1513-1529, 1999). Furthermore, Mre11, along with Rad50 and Xrs2/p95, plays a critical role in the DNA damage response, as well as G2/M cell arrest following DNA damage, and DNA repair (Dolganov G M et al.,
Mol Cell Biol.
16: 4832-4841, 1996; Carney J P et al.,
Cell
93: 477-486, 1998; Lee S E, et al.,
Cell
94: 399-409, 1998). Recently, Mre11 has been shown to be essential for the maintenance of chromosomal DNA (Yamaguchi-Iwai Y et al.,
Embo J.
18: 6619-6629, 1999).
In summary, MRE11 is an important gene involved in meiotic and mitotic recombination, as well as homologous and non-homologous recombination. Thus, this single protein participates in multiple pathways that are often competing with each other such as double-strand break (DSB) formation in meiosis and DSB repair (via non-homologous end joining pathway) in mitosis. A very recent study by Furuse M, et al. employed two specific mutants of yeast Mre11 to elucidate this phenomenon (Furuse M, et al.
EMBO J.
17: 6412-6425; 1998). A point mutation in Mre11 (Asp16Ala) completely abolished the nuclease activity, without any change in DNA binding activity. This mutation also conferred MMS sensitivity to mitotic cells and caused them to accumulate unprocessed DSBs during meiosis. However, another mutant carrying a deletion of 49 C-terminal amino acids had almost wild-type levels of nuclease activity but reduced DNA binding activity. The mitotic phenotypes of this mutant were essentially unchanged, but the meiotic DSB formation was reduced dramatically. These results indicate the presence of two distinct functional domains on the Mre11 protein, an N-terminal region specifically involved in mitotic functions and a C-terminal 49 amino acid domain involved in the meiotic DSB formation. Thus, interactions of different domains with other proteins (such as Rad50 and Xrs2/P95) may be an underlying mechanism for the distinct roles of Mre11 in meiosis and mitosis (Usui T et al.,
Cell
95: 705-716, 1998). Whatever mechanisms may be involved, it is clear that either null or the N-terminal nuclease domain mutants of Mre11 are deficient in non-homologous end-joining.
Homologues of yeast MRE11 have been isolated from
S. pombe
(Tavassoli M et al.,
Nucleic Acid Res.
23: 383-388, 1995), human (Petrini J H et al.,
Genomics
29: 80-86, 1995; Chamankhah M et al.,
Gene
225: 107-116, 1998), and mouse (Xiao Y and Weaver D,
Nucleic Acid Res.
25: 2985-2991, 1997). Similarly, cDNA sequences encoding yeast Mre11-like proteins from Drosophila (Accession No. AF132144) Xenopus (Accession No. AF134569), Coprinus (Accession No. AF178433) and Arabidopsis (Accession No. AJ243822) have been deposited in the Genbank database.
Control of non-homologous end joining as well as mitotic and meiotic recombination by the modulation of Mre11, provides the means to modulate the efficiency with which heterologous nucleic acids are incorporated into the genomes of a target plant cell. Control of these processes has important implications in the creation of novel recombinantly engineered crops such as maize. The present invention provides this and other advantages.
SUMMARY OF THE INVENTION
The present invention teaches a full-length cDNA for a Mre11 orthologue. The protein shares homology with the published Mre11 sequences. For example, the N-terminal Asp16 residue from the yeast Mre11 sequence which is involved in nuclease function is conserved in the maize protein as are several motifs found in many members of the phosphodiesterase/Mre11 gene family (Example 4). Generally, it is the object of the present invention to provide nucleic acids and proteins relating to Mre11. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention, and methods for modulating, in a transgenic plant, expression of the nucleic acids of the present invention. It is also an object of the present invention to provide methods for increasing transformation efficiency.
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 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 host cell into which has been introduced the recombinant expression cassette, and 3) a transgenic plant comprising the recombinant expression cassette. The host cell and plant are optionally from maize, wheat, rice, or soybean. The present invention also provides transgenic seed from the transgenic plant.
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 a further aspect, the present invention relates to a polynucleotide amplified from a
Zea mays
nucleic acid library using primers which selectively hybridize, under stringent hybridization conditions, to loci within polynucleotides of the present invention.
In another aspect, the present invention relates to a

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