Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-01
2002-05-28
Wang, Andrew (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S091100, C435S468000, C536S023100, C800S278000, C800S298000
Reexamination Certificate
active
06395892
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid molecules isolated from Populus species, and methods of using these molecules and derivatives thereof to produce plants, particularly trees such as Populus species, that have modified fertility characteristics.
BACKGROUND OF THE INVENTION
The increasing demand for pulp and paper products and the diminishing availability of productive forest lands are being addressed in part by efforts to develop trees that produce increased yields in shorter growth periods. Many such efforts are focused on the production of transgenic trees having modified growth characteristics, such as reduced lignin content (see for example, U.S. Pat. No. 5,451,514, “Modification of Lignin Synthesis in Plants”), and resistance to insect, viruses and herbicides. A major concern with the production of transgenic trees is the possibility that the transgenic traits might be introduced into indigenous tree populations by cross-fertilization. Thus, for example, the introduction of genes for insect resistance into indigenous tree populations could accelerate the evolution of resistant insects, adversely affect endangered insect species and interfere with normal food chains. Because of these concerns, the U.S. and other governments have instituted regulatory review processes to assess the risks associated with proposed environmental releases of transgenic plants (both for field trials and commercial production).
Genetic engineering of sterility into trees offers the possibility of securing introduced genes in the engineered tree; trees that produce neither pollen nor seeds will not be able to transmit introduced genes by normal routes of reproduction. Additional potential benefits of engineering sterility into trees include increased wood yields and reduced production of allergens such as pollen. For a review of engineering reproductive sterility in forest trees, see Strauss et al. (1995a,b).
Two primary methods for engineering sterility have been described. In the first method, termed genetic ablation, a cytotoxic gene is expressed under the control of a reproductive tissue-specific promoter. Cytotoxic genes employed in this method to date include RNase (Mariani et al., 1990; Mariani et al., 1992; Reynarts et al., 1993; Goldman et al., 1994), ADP-ribosyl transferase (Thorsness et al., 1991; Kandasamy, 1993; Thorseness et al., 1993), the Agrobacterium RoiC gene (Schmüilling, 1993), and glucanase (Worrall et al., 1992, Paul et al., 1992). The expression of the cytotoxic gene results (ideally) in the death of all cells in which the reproductive tissue-specific promoter is active. It is therefore critical that the promoter be highly specific to the reproductive tissue to avoid pleiotropic effects on vegetative tissue. For this reason, genome position effects on the transgene need to be monitored (see Strauss et al., 1995a,b). The success of genetic ablation methods in trees will thus depend on the availability of a suitable reproductive tissue-specific promoter for the tree species in question.
The second method for engineering sterility involves inhibiting the expression of genes that are essential for reproduction. This can be accomplished in a number of ways, including the use of antisense RNA, sense suppression and promoter-based suppression. Details and applications of antisense (Kooter, 1993; Mol et al., 1994; Van der Meer et al., 1992; Pnueli et al., 1994), sense suppression (Flavell, 1994; Jorgensen, 1992; Taylor et al., 1992) and promoter-based suppression (Brusslan et al., 1993; Matzke et al., 1993) technologies in plants have been described in the scientific literature. The key to the use of any of these methods in the production of sterile trees is the identification of appropriate indigenous genes, i.e, disruption of the expression of such genes must result in the abolition of correct reproductive tissue development.
Genes specifically expressed in reproductive tissues have been isolated from a number of plant species (for a review, see Strauss et al., 1995a). Genes that have been characterized as acting early in the development of floral structures include LEAFY (LFY) from Arabidopsis (Weigel et al., 1992), APETALAI (AP1) from Arabidopsis (Mandel et al, 1992a,b), and FLORICAULA (FLO) from Antirrhinum (Coen et al., 1990), which regulate the transition from inflorescence to floral meristems. APETALA2 (AP2) appears to regulate the AGAMOUS gene (AG) which plays a role in differentiation of male and female floral tissues (see Okamuro et al., 1993). DEFICIENS (DEF) is a floral homeotic gene from Antirrhinum that is expressed throughout flower development (Schwarz-Sommer et al. 1992).
The majority of floral homeotic genes are members of the MADS-box family of transcription factors (Yanofsky et al., 1990). The MADS-box is a conserved region of approximately 60 amino acid residues. MADS is an acronym for the first four known genes in which the MADS-box was identified: yeast minichromosomal maintenance factor (MCM1), the floral homeotic genes AG and DEF, and human serum response factor (SRF). Plant MADS-box genes contain four domains: the highly conserved MADS-box region located near or at the 5′ end of the translated region in plant genes; the L or linker region between the MADS and K domains; the K domain, a moderately conserved keratin-like region predicted to form amphipathic &agr;-helices; and a highly variable carboxy-terminal region. The K-box is only present in plant MADS-box genes. It is thought to be involved in protein-protein interactions (Pnueli et al., 1991).
Studies have shown that the organization of the MADS domain in plants is similar to that in SRF; the basic N-terminal portion of the domain is required for DNA-binding and the C-terminal half of the box is required for dimerization. Because MADS proteins bind DNA as dimers, the MADS box as well as a C-terminal extension that is involved in dimerization are required for DNA-binding. The C-terminal extension varies throughout the gene family. C-terminal deletions indicate that the minimal DNA-binding domain of AP1 and AG includes the MADS-box and part of the L region, whereas AP3 and PI require a portion of the K box in addition to the MADS and L regions (Riechmann et al., 1996). The difference in the sizes of the minimal binding domains is thought to reflect the dimerization characteristics of the respective proteins: AP1 and AG bind DNA as homodimers whereas AP3/PI and their Antirrhinum homologs DEF/GLO bind as heterodimers.
MADS-box proteins have been found to bind to a motif found in target gene promoters referred to as the CArG-box. CArG-box motifs are also found in the promoters of MADS-box genes, where they are thought to be targets for auto-regulation. Riechmann et al. (1996) used circular permutation and phasing analysis to detect conformational changes in DNA that resulted from MADS-box protein binding (Reichmann et al., 1996). They found that bound AP1, AP3/PI, and AG all induce DNA bending oriented toward the minor groove. For a review of MADS box biology, see Ma, 1994; Purugganan et al., 1995; and Yanofsky, 1995. AG and DEF have been characterized as MADS box genes; while FLO and LFY appear to encode transcription factors and have proline-rich and acidic domains, they are not MADS box genes.
Following a functional analyses of MADS box genes, Mizukami et al. (1996) created deletion mutants of AG in which various domains of the gene, including the MADS and K boxes were deleted. Based on their results, they proposed that dominant negative mutations of MADS box genes could be created by deleting the all or part of the MADS domain, or by deleting all or part of the K domain or by deleting various portions of the 3′ region of the AG open reading frame. It was proposed that the proteins encoded by these deletion mutants would be able to bind either the target DNA (i.e., the nucleotide sequence to which the transcription factor binds) or the protein co-factors required for transcription, but not both. Thus, it was proposed that such mutant proteins would interfere with
Brunner Amy
Rottmann William
Sheppard Lorraine
Strauss Steven H.
Klarquist & Sparkman, LLP
The State of Oregon Acting by and through the State Board of Hig
Wang Andrew
Zara Jane
LandOfFree
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