Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1998-11-06
2002-11-19
Fox, David T. (Department: 1638)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C435S069100, C435S252330, C435S468000, C536S023600
Reexamination Certificate
active
06482646
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding proteins that interact with nuclear matrix proteins and function as transcriptional activators.
BACKGROUND OF THE INVENTION
The nuclear matrix hypothesis proposes a structural framework for the eukaryotic nucleus that is similar to the cytoskeleton. To date, its best characterized component is the lamina, a filamentous protein network that lines the inner membrane of the nuclear envelope. Major components of the lamina include a group of intermediate-filament (IF) proteins, collectively known as nuclear lamins, that are classified as type A, B, and C (McKeon et al.,
Nature
319:463-468 (1986)). Lamin B is attached to the inner nuclear membrane via a C-terminal C15 farnesyl group (Schafer et al.,
Annu. Rev. Genet.
30:209-237 (1992)), whereas lamins A and C bind to lamin B. Other integral membrane proteins interact with lamin B and most likely stabilize the membrane attachment of lamins (Furukawa et al.,
EMBO J.
14:1626-1636 (1995)). Recent studies have also demonstrated the ability of lamins A and B to bind DNA, suggesting a role for mammalian lamins in anchoring chromatin to the nuclear envelope. The interaction between nuclear envelope, lamina, and chromatin is considered to be of fundamental importance for higher order chromosome organization, as well as the assembly and disassembly of the nuclear envelope during mitosis (Furukawa et al.,
EMBO J.
14:1626-1636 (1995)).
The nuclear matrix is a second structural skeleton that has been biochemically defined as the insoluble component that remains after treatment of isolated nuclei with DNase I and extraction of proteins with high-salt solutions (Berezney et al.,
Biochem. Biophys. Res. Comm.
60:1410-1417 (1974)) or the chaotropic agent lithium diiodosalicylate (Mirkowitch et al.,
Cell
39:223-232 (1984)). Chromatin binds to the nuclear matrix via matrix attachment regions (MARs) in the DNA. MARs are generally AT-rich DNA sequences that are several hundred base pairs long and localized to noncoding regions of the DNA, but often flanking genes (Gasser et al.,
Trends Genet.
3:16-22 (1987)). However, there is no consensus sequence known for MARS. The significance of structural characteristics for MARs such as DNA bending and a narrow minor groove due to oligo(dA) tracts has been previously proposed. MARs have been shown to increase transcriptional activity of a linked gene and to confer position-independent, copy-number dependent expression in stably transfected cells (Phi-Wan et al.,
EMBO J.
7:655-664 (1988)).
A small number of MAR binding proteins have been identified from animal nuclei, and they are considered to be components of the nuclear matrix (von Kries et al.,
Cell
64:123-135 (1991); Dickinson et al.,
Cell
70:631-645 (1 992); Romig et al.,
EMBO J.
11:3431-3440 (1992); Tsutsui et al.,
J. Biol. Chem.
268:12886-12894 (1993); Renz et al.,
Nucleic Acids Res.
24:843-849 (1996); U.S. Pat. No. 5,652,340). In addition, it has been shown that lamins specifically bind to MARs (Luderus et al.,
Mol. Cell. Biol.
14:6297-6305 (1994)). The specific interaction between DNA and the nuclear matrix
uclear lamina is most likely an important mechanism for long-range gene regulation and higher order chromatin organization (Gasser et al.,
Trends Genet.
3:16-22 (1987)).
Most investigations into structural components of the nucleus have focused on proteins in vertebrates and Drosophila. Significantly less information is available for other eukaryotes, and in particular for plants. Proteins that are immunologically related to animal IF proteins and lamins have been detected in pea and carrot nuclei (Beven et al.,
J. Mol. Biol.
228:41-57 (1991); McNulty et al.,
J. Cell Sci.
103:407-414 (1992)). Plant nuclear matrix preparations that bind to animal MARs have been reported, suggesting that proteins with similar DNA binding specificities exist in plants as well (Hall et al.,
Proc. Natl. Acad. Sci. USA
88:9320-9324 (1991)).
Effects of MARs on gene expression in plants have been reported, but have been quite variable. In some experimental systems, no reduction of variability but an increase in expression level has been reported (Breyne et al.,
Plant Cell
4:463-471 (1992); Allen et al.,
Plant Cell
5:603-613 (1993); Allen et al.,
Plant Cell
8:899-913 (1996); U.S. Pat. No. 5,773,689). Other authors have found no significant increase in expression level, but a reduction of variability (van der Geest et al.,
Plant J.
6:413-423 (1994); Mlynarova et al.,
Plant Cell
6:417-426 (1994)). It is not clear what causes these observed differences, but they will most probably be due to the fact that MARs establish different molecular interactions, which might either depend on the features of the MAR itself or on the specific molecular environment of the transformed cell/tissue. The routine use of MARs for strategies to improve transgene expression will greatly depend on the characterization of the proteins involved in DNA-nuclear matrix attachment and the factors responsible for the observed increase in gene expression.
Currently, no sequence information is available for plant lamin-like proteins. However, the cloning of the cDNA for a plant MAR-binding protein, MFP1, from tomato has been reported (Meier et al.,
Plant Cell
8:2105-2115 (1996)). MFP1 has structural features of a filament-like protein and it preferentially binds to MAR DNA sequences from both plants and animals. In contrast to other known MAR binding proteins, MFP1 contains a hydrophobic N-terminal amino acid sequence that might function as a membrane-spanning domain. MFP1, therefore, has features of a novel anchor protein that most likely connects chromatin via MAR DNA with the nuclear envelope and nuclear filament proteins.
In order to routinely use the attachment of transgenes to the nuclear matrix improve gene expression, it will be necessary to further characterize the elements involved in this process and to better understand the underlying mechanisms. Thus, a need exists to identify and characterize additional nuclear matrix proteins. The present invention presents six previously unknown proteins that are localized in the nuclear matrix, bind to a MAR-binding protein or to a protein that binds to a MAR-binding protein, or are able to increase gene expression.
SUMMARY OF THE INVENTION
Applicants provide a method for regulating gene expression in a stably transformed transgenic plant cell which comprises combining into the genome of the plant cell:
(a) a first chimeric gene comprising in the 5′ to 3′ direction:
(1) a promoter operably-linked to at least one DNA-binding domain sequence;
(2) a coding sequence or a complement thereof operably-linked to the promoter; and
(3) a polyadenylation signal sequence operably-linked to the coding sequence or a complement thereof;
provided that when the promoter is a minimal promoter then the DNA-binding domain sequence is located upstream of the minimal promoter; and
(b) a second chimeric gene comprising in the 5′ to 3′ direction:
(1) a promoter;
(2) a DNA sequence encoding a DNA-binding domain;
(3) a DNA sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:14 operably-linked to the DNA sequence of (2); and
(4) a polyadenylation signal sequence operably-linked to the DNA sequence of (3),
wherein the expression of the second chimeric gene regulates expression of the first chimeric gene.
Applicants also provide a further method for regulating gene expression in a stably transformed transgenic plant cell which comprises (a) transforming the genome of the plant cell with:
(1) a chimeric gene comprising in the 5 ′ to 3′ direction:
(i) a promoter operably-linked to at least one DNA-binding domain sequence;
(ii) a coding sequence or a complement thereof operably-linked to the promoter; and
(iii) a polyadenylation signal sequence operably-linked to the coding sequence or a complement thereof;
provided that when the
Gindullis Frank
Meier Iris
E. I. du Pont de Nemours and Company
Fox David T.
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