Matrix attachment regions

Details Matrix attachment regions Matrix attachment regions

2000-04-21

2001-01-23

Guzo, David (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S320100, C435S468000, C435S410000, C435S430000, C800S295000, C800S298000, C536S023100, C536S023600, C536S024100

Reexamination Certificate

active

06177612

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to nucleic acid molecules isolated from the 5′ flanking region of endosperm-specific storage protein genes of monocotyledonous plants, and which possess nuclear matrix binding activity.
2. Description of the Related Art
In the past fifteen years it has become possible to transfer genes from any organism into a wide range of crop plants including the major monocotyledonous cereal crops wheat, rice, barley, oat and maize. However, even though the introduced genes can be expressed in the transformed crops, the level of expression can be very low. Indeed, Peach and Velten (1991) found that the majority of detectable transformants exhibited very low expression. The variation in expression is due to the influence of the surrounding chromatin at the site of insertion of the transgene (position effects). As a result, large numbers of transgenic plants must be produced in order to be sure of producing a single high-expressing plant. This is not trivial in cereal crops where the transformation efficiency is only 1 to 5%. In addition to expression variability, there is also the possibility that transgene silencing will occur in subsequent generations.
For the purposes of crop improvement, it would be highly beneficial to reduce the numbers of transgenic plants which need to be produced to find a high-expressing plant, and also to ensure that there will be no transgene silencing in subsequent generations.
In eukaryotes, the DNA is folded into chromosomes in the form of loops. These loops are anchored to a proteinaceous nucleoskeleton, known as the nuclear matrix or scaffold, by segments of DNA known as matrix attachment regions (“MAR”s). The DNA loop structure, which allows for the unwinding of DNA to permit access by transcriptional regulatory proteins, has important implications for gene regulation and expression. One function of the loop structure and matrix attachment regions is to insulate genes from the effects of surrounding chromatin, thereby allowing copy number dependent, position independent expression of adjacent genes. For this reason, various studies have investigated the hypothesis that MARs may allow position independent expression of any introduced genes in transgenic plants.
In several studies, the hypothesis was validated, and MARs were found to increase transgene expression, decrease silencing, prevent silencing after crossing or backcrossing to non-transformed plants, normalize expression, and to provide copy number independent expression (Allen et al. (1996); Allen et al. (1993); Ulker et al. (1997); Mlynarova et al. (1995); Breyne et al. (1992)). These studies utilized MARs derived from either animal or tobacco sources.
Recently, MARs from other plant species such as maize and rice have been isolated (Avramova and Bennetzen (1993); Nomura et al.(1997)). The maize MAR was derived from the promoter of an alcohol dehydrogenase gene. The rice MARs were isolated on the basis of functional binding to the nuclear matrix, and their association with any transcribed gene is unknown.
All of the MARs isolated to date have similar features in that: (1) they are AT rich (greater than 60%); (2) they contain motifs such as A-boxes (consensus sequence AATAAA(T/C)AAA—SEQ ID NO: 6, T-boxes (consensus sequence TT(T/A)T(T/A)TT(T/A)TT—SEQ ID NO: 7), ATATTT boxes, and boxes showing homology to DNA topoisomerase II consensus cleavage sites (consensus sequence GTN(A/T)A(T/C)ATTNATNN(G/A)—SEQ ID NO: 8); and (3) they usually have a length of about 300 to 500 nucleotides (see e.g. Breyne et al. (1992)). None of the mentioned motifs are universal, however, and different MARs do not have extensive homology, nor are there strictly conserved DNA elements. Specific MAR DNA sequences are therefore species specific and differ in their ability to bind the nuclear matrix. Each putative MAR must therefore be investigated by functional assays.
Given the low transformation efficiency in cereal crop plants, MARs functional in cereal crop plants to increase transgene expression, decrease silencing, prevent silencing after crossing or backcrossing to non-transformed plants, normalize expression, and to provide copy number independent expression, would be very useful. The species-specificity of MARs limits the utility of the known rice and maize MARs for improving transgene expression in monocotyledonous cereal crops such as wheat (
Triticum aestivum
). Thus, there is an ongoing need for MARs functional in cereal crop plants.
SUMMARY OF THE INVENTION
The present invention provides a DNA sequence, designated “MAR Bx7”, isolated from a region adjacent to a wheat storage protein promoter. The DNA sequence, which is depicted in SEQ ID NO: 1, has features characteristic of MARs in that it is AT rich (63%), and contains motifs homologous to the DNA topoisomerase II consensus sequence (position 709), T-box (position 118) and AATATATTT motif (position 437). Proof that this sequence acts as a matrix attachment region was provided by a functional test of binding to the nuclear matrix. The sequence binds the nuclear matrix and binds more strongly than a previously isolated MAR from maize. The sequence has also been demonstrated to have no deleterious effect when placed adjacent to a heterologous promoter, indicating its utility in enhancing and stabilizing transgene expression. Therefore, in one aspect, the invention provides an isolated nucleic acid molecule comprising the nucleic acid sequence depicted in SEQ ID NO: 1.
As stated above, MAR Bx7 was isolated from the 5′ flanking region of a wheat storage protein gene, specifically the Bx7 storage protein gene from
T. aestivum
variety Glenlea. In the developing endosperm of cereals, storage proteins are produced which are not found in any other plant part or at any other stage of development. The genes encoding these proteins are very highly expressed, with up to two percent of the seed protein attributable to each gene. The genes are regulated at the transcriptional level, and are very tightly regulated in that they are only expressed in the developing endosperm. The tight regulation and high expression of these endosperm-specific storage protein genes, including the Bx7 storage protein gene, indicates that MAR Bx7 is highly effective at allowing very high expression rates, and would therefore be useful in enhancing the expression of transgenes in transformed plants.
The present invention provides methods by which the exemplified MAR sequence can be used to identify and isolate other MAR sequences from the 5′ flanking region of endosperm-specific storage protein genes of monocotyledonous plants. The inventors have identified DNA sequences within the 5′ flanking regions of other endosperm-specific storage protein genes that are closely homologous to the nucleotide sequence of MAR Bx7. The invention provides methods whereby these additional putative MAR sequences can be assayed for nuclear matrix binding activity, and for their ability to enhance or stabilize transgene expression in transgenic monocotyledonous plants. Such additional MAR sequences of the invention have a length of at least 300 nucleotides, preferably at least 500 nucleotides. For use in transforming monocotyledonous plants with a transgene, the MAR sequences of the invention are preferably operably linked to at least one DNA construct which includes a plant promoter, a coding sequence for the gene to be expressed in the plant, and a poly(A) addition signal. Preferably, a MAR sequence of the invention is operably linked both upstream and downstream of the DNA construct. However, a single MAR sequence either upstream or downstream of the DNA construct is sufficient to confer the benefit of the presence of the MAR sequence on expression of the transgene. Therefore, in another aspect, the invention provides a recombinant nucleic acid molecule comprising: a matrix attachment region comprising a nucleic acid molecule having a length of at least 300 nucleotides and which possesses nuclear matrix binding activit

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