Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1998-07-24
2001-06-12
LeGuyader, John L. (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S298000, C800S314000, C800S317000, C435S320100, C435S325000, C435S410000, C435S414000, C435S419000, C536S023100, C536S024100
Reexamination Certificate
active
06245974
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to matrix attachment regions isolated from a higher plant, and to methods for isolating matrix attachment sequences.
BACKGROUND OF THE INVENTION
The proteinaceous nuclear ‘matrix’ or ‘scaffold’ in the cell nucleus plays a role in determining chromatin structure. Electron micrographs show that nuclear DNA is attached to this scaffold at intervals to produce a series of loops (Zlatanova and Van Holde,
J. Cell Sci.
103:889 (1992)). Matrix Attachment Regions (MARs; also referred to a scaffold attachment regions or SARs) are genomic DNA sequences which bind specifically to components of the nuclear matrix. See Boulikas,
J. Cell. Biochem.
52:14 (1993). These sequences are thought to define independent chromatin domains through their attachment to the-nuclear matrix. Both transcription and replication are thought to occur at the nuclear matrix.
Transformation of a cell using a; transgene flanked by one or more MARs has been shown to increase expression of the transgene product, compared to transformation using a construct lacking MARs. See Allen et al.,
Plant Cell
8:899 (1996); Bonifer et al.,
EMBO J.
9:2843 (1990); McKnight et al.,
Proc. Natl. Acad. Sci. USA
89:6943 (1992); Phi-Van et al.,
Mol. Cell. Biol.
10:2303 (1990)). Flanking a GUS reporter gene with yeast MARs has been reported to result in higher and less variable transgene expression in plant cells. Allen et al.
Plant Cell
5:603 (1993).
SUMMARY OF THE INVENTION
In view of the foregoing, a first aspect of the present invention is an isolated DNA molecule having a nucleotide sequence selected from the group consisting of SEQ ID NOS:1, 2, 4-11 and 13, sequences that hybridize to this isolated DNA under stringent conditions.
A further aspect of the present invention is a DNA construct comprising a transcription initiation region, a structural gene operatively associated with the transcription initiation region, and at least one matrix attachment region of the present invention positioned either 5′ to the transcription initiation region or 3′ to the structural gene.
A further aspect of the present invention is a vector comprising a DNA construct as described above, including plasmids, viruses and plant transformation.
A further aspect of the present invention is a host cell containing a DNA construct as described above, including plant and animal host cells.
A further aspect of the present invention is a method of identifying matrix attachment regions in a DNA molecule of known nucleotide sequence, by identifying a sequence section of at least twenty contiguous nucleotides that is at least 90% A or T nucleotides. The method may further comprise preparing a MAR molecule of at least about 300 nucleotides, comprising the identified MAR motif.
REFERENCES:
patent: WO 94 07902 (1994-04-01), None
patent: WO 97 27207 (1997-07-01), None
Allen et al.; High-Level Transgene Expression in Plant Cells: Effect of a Strong Scaffold Attachment Region from Tobacco,The Plant Cell:8:899-913 (1996).
Journal of Cellular Biochemistry, Keystone Symposia on Molecular & Cellular Biology, Supp 21B:167 (1995).
Abstracts—4 th International Congress of Plant Molecular Biology, Amsterdam, Jun. 19-24, 1994.
Boulikas et al.; A novel class of matrix attached regions (MARs) identified by random cloning and their implications in differentiation and carcinogenesis,Int'l Journal of Oncology:2:325-330 (1993).
Allen et al.; Scaffold Attachment Regions Increase Reporter Gene Expression in Stably Transformed Plant Cells,The Plant Cell:5:603-613 (1993).
Brylawski et al.; Association of Putative Origins of Replication with the Nuclear Matrix in Normal Human Fibroblasts1,Cancer Research:53:3865-3868 (1993).
Forrester et al.; Dependence of Enhancer-Mediated Transcription of the Immunoglobulin &PHgr; Gene on Nuclear Matrix Attachment Regions,Science:265:1221-1225 (1994).
Jarman et al.; Nuclear scaffold attachment sites in the human globin gene complexes,The EMBO Journal:vol. 7:11:3337-3344 (1988).
Kas et al.; Anchorage of the Chinese Hamster Dihydrofolate Reductase Gene to the Nuclear Scaffold Occurs in an Intragenic Region,J. Mol. Biol.:198:677-692 (1987).
Geest et al.; The &bgr;-phaseolin gene is flanked by matrix attachment regions,The Plant Journal:vol 6(3):413-423 (1994).
Cockerill et al.; Chromosomal Loop Anchorage of the Kappa Immunoglobulin Gene Occurs next to the Enhancer in a Region Containing Topoisomerase II Sites,Cell:44:273-282 (1986).
Hall et al.; Nuclear scaffolds and scaffold-attachment regions in higher plants,Proc. Natl. Acad. Sci. USA:88:9320-9324 (1991).
Hall et al.; Isolation and characterization of nuclear scaffold,Plant Molecular Biol. Manual:D2:1-12 (1994).
Phi-Van et al.; The matrix attachment regions of the chicken Iysozyme gene co-map with the boundaries of the chromatin domain,The EMBO Journal:vol 7(3):655-664 (1988).
Levy-Wilson et al.; The Limits of the Dnase I-sensitive Domain of the Human Apolipoprotein B Gene coincide with Locations of Chromosomal Anchorage Loops and Define the 5′ and 3′ Boundaries of the Gene,The J. of Biol. Chemistry:264(35):21196-21204 (1989).
Gasser et al.; Cohabitation of Scaffold Binding Regions with Upstream/Enhancer Elements of Three Developmentally Regulated Genes of D. melanogaster,Cell:46:521-530 (1986).
Michalowski Susan
Spiker Steven
LeGuyader John L.
Myers Bigel & Sibley & Sajovec
North Carolina State University
Shibuya Mark L.
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