Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Nonplant protein is expressed from the polynucleotide
Reexamination Certificate
2000-12-08
2004-05-11
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Nonplant protein is expressed from the polynucleotide
C435S320100, C435S419000, C435S468000, C435S252300, C435S200000, C536S023200, C536S023740, C800S293000, C800S294000
Reexamination Certificate
active
06734344
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to a &bgr;-(1,3) exoglucanase gene of
Coniothyrium minitans.
BACKGROUND OF THE INVENTION
The plant cell wall provides stability, protects against pathogens, and influences the growth and development of the plant cell, among other functions. Structurally, the plant cell wall consists of a primary and a secondary wall, both containing cellulose microfibrils embedded in a matrix of carbohydrates (specifically polysaccharides), structural glycoproteins, enzymes, and other components. Carbohydrate polymers have been well characterized and play a primary role in maintaining the structural rigidity of the plant cell wall. In this regard, the plant cell wall sequesters significant amounts of metabolically inactive polysaccharides from among the following classes:
i) celluloses (insoluble fibrils of &bgr;-(1,4) glucans);
ii) hemi-celluloses (non-cellulosic polysaccharides which include &bgr;-(1,3) glucans, &bgr;-(1,3))(1,4) glucans, mannans, and xylans); and
iii) lignin (a polyphenolic compound) (Thomson, 1993).
The &bgr;-glucans are polymers of glucose molecules formed by &bgr;-links between the glucose molecules. The links may be &bgr;-(1,4), &bgr;-(1,3), or &bgr;-(1,6) or a mixture of those in such polymers. &bgr;-glucans are ubiquitous in the natural flora. Many classes of &bgr;-glucan polymers exist, and their chemical structure, physiological function, and predominance differ among plant and fungal species.
A. Cellulosic &bgr;-Glucans
Cellulosic &bgr;-(1,4) glucans are polymeric chains formed by successive glucose monomers covalently joined by &bgr;-(1,4) glucan linkages. These &bgr;-(1,4) glucan chains associate In bundles to form rigid, insoluble microfibrils which may contain up to several hundred cellulosic polymers (Beguin and Aubert, 1994). The tensile strength of such cellulose microfibrils in the plant cell wall selves to confer rigidity to plant structures. Further, cellulosic components, together with other polymeric compounds in the plant cell wall, demonstrate a protective role by acting as a barrier to various phytopathogens.
B. Non-cellulosic &bgr;-Glucans
While cellulosic polymers are ubiquitous in the cell walls of diverse plant species, non-cellulosic glucans (&bgr;-(1,3) glucans and &bgr;-(1,3)(1,4) glucans) are typically present in the cell walls of some monocotyledonous plant families, such as the Poaceae (Gramineae) (Chesson et al., 1995). In fungi, non-cellulosic &bgr;-(1,3) glucans are predominant in the cell wall, notably providing structural resilience (Borgia and Dodge, 1992). In addition to providing structural stability to the fungal cell wall, &bgr;-(1,3) glucans serve as carbohydrate reserves in nutritionally-depleted growth environments (Copa-Patino et al., 1989).
The hydrolysis of non-cellulosic &bgr;-glucans by &bgr;-glucanase enzymes is of great significance to plant-mycopathogen interactions, fungal cell wall architecture, and forage feed digestion in ruminants (Umemoto et al., 1997; Vasquez-Garciduenas et al., 1998; Chen et al., 1997). Such enzymes have been classified into different families according to their origin (plant, fungal, or microbial), substrate specificity, and function (Table 1). Different non-cellulosic &bgr;-glucanases thus have distinct substrates and modes of hydrolytic action, to the extent that plant, fungal, and microbial non-cellulosic &bgr;-glucanases each belong to specific families with conserved sequence and functional properties.
i) Non-Cellulosic &bgr;-Glucanases in Plants
In plants, non-cellulosic glucanases may be classified as either &bgr;-(1,3) endoglucanases (laminarinases) or &bgr;-(1,3)(1,4) glucanases (mixed linkage glucanases or lichenases) according to substrate specificity and function (Table 1). &bgr;-(1,3) endoglucanases (EC 3.2.1.39) hydrolyze successive &bgr;-(1,3) glucan (laminarin) chains in an endoglucanase manner (i.e. random digestion within the polymeric chain), whereas &bgr;-(1,3)(1,4) glucanases (EC 3.2.1.73) specifically degrade mixed-linkage glucans (non-cellulosic glucans containing glycosidic &bgr;-(1,3) and &bgr;-(1,4) linkages such as lichenan) by hydrolyzing a &bgr;-(1,4) linkage adjacent to a &bgr;-(1,3) linkage in the same manner (Hoj and Fincher, 1995).
In addition to targeting different substrates, &bgr;-(1,3) endoglucanases and &bgr;-(1,3)(1,4) glucanases are distinct functionally. &bgr;-(1,3) endoglucanases appear to comprise a large family of pathogenesis-related proteins produced by plants during infection by pathogens. During the plant-pathogen interaction between soybean plants (Glycine max) and the fungal pathogen
Phytophthora megaspora
f. sp. glycinea, soybean &bgr;-(1,3) endoglucanases are able to digest the fungal cell walls (Umemoto et al., 1997). The liberated fungal &bgr;-(1,3) oligoglucans subsequently bind a &bgr;-oligoglucan receptor in the plant cell membrane, initiating a signal transduction event, and ultimately stimulating plant defense responses such as phytoalexin accumulation. &bgr;-(1,3) endoglucanases thus appear to weaken and degrade fungal cell walls, while liberating elicitor compounds (such as &bgr;-oligoglucan) in order to upregulate plant defense responses.
In comparison, &bgr;-(1,3)(1,4) glucanases may play an important role in nutrient mobilization during seed germination in some plant species. During barley (
Hordeum vulgare
) seed germination, the &bgr;-(1,3)(1,4) glucanases degrade the &bgr;-(1,3)(1,4) glucan-rich cell wall in the seed endosperm, allowing the diffusion of amylases and proteases into starch and protein stores in the endosperm compartment (Hoj and Fincher, 1995).
Although &bgr;-(1,3) endoglucanases thus differ functionally from &bgr;-(1,3)(1,4) glucanases, these glucanase types in plants are structurally conserved, appearing to originate from a common ancestor (Hoj and Fincher, 1995).
ii) Non-Cellulosic &bgr;-Glucanases in Fungi
In comparison to &bgr;-(1,3) endoglucanases and &bgr;-(1,3)(1,4) glucanases in plants, fungal glucanases differ in both sequence and function (Table 1). In fungi, non-cellulosic glucanases consist of the following classes: &bgr;-(1,3) exoglucanase (EC 3.2.1.58); &bgr;-(1,3) endoglucanase (EC 3.2.1.39); &bgr;-(1,3)(1,4) endoglucanase (EC 3.2.1.73); and &bgr;-(1,3)/(1,3)(1,4) glucanase (EC 3.2.1.6). Fungal &bgr;-(1,3) exoglucanases are quintessential enzymes in mycoparasitism. Mycoparasites, such as
Trichoderma hazarium
, rely on &bgr;-(1,3) exoglucanases to hydrolyze the cell wall of various fungal phytopathogens, thus liberating nutritionally available oligoglucans for absorption and metabolism (Vasquez-Garciduenas et al., 1998). Further, fungal &bgr;-(1,3) exoglucanases have been implicated in the autolysis of fungal cell walls in nutritionally-depleted environments (Copa-Patino et al., 1989; Stahmann et al., 1993). In addition, &bgr;-(1,3) exoglucanases may have a morphogenic role in fungal growth and differentiation (Peberdy, 1990).
The prevalence of &bgr;-(1,3)(1,4) endoglucanases in fungi has yet to be confirmed. To date, few of these have been cloned, with the pioneering example being a mixed-linkage glucanase from the ruminal anaerobic fungus Orpinomyces (licA) (Chen et al., 1997). Such mixed-linkage glucanases from ruminal organisms are presumably produced to improve the digestibility of non-cellulosic &bgr;-glucans from fibrous forage feed.
iii) Non-Cellulosic &bgr;-Glucanases in Bacteria
In bacteria, non-cellulosic glucanases consist of &bgr;-(1,3)(1,4) glucanases (EC 3.2.1.73), which are specific for the substrate, &bgr;-(1,3)(1,4) glucan (Table 1). Examples of such microbial glucanases include enzymes from ruminal and non-ruminal microbial species (e.g.
Fibrobacter succinogenes
and
Bacillus subtilis
respectively) (Teather and Erfle, 1990; Wolf et al., 1995).
iv) Non-Cellulosic &bgr;-Glucanases in Lower Animalia
A metazoan &bgr;-(1,3) endoglucanase from the sea urchin
Strongylocentrotus purpuratus
has been characterized, apparently having a bacterial origin (Bachman and McClay, 1996). Its presence in sea urchin eggs implies that the enzyme
Cheng Kuo Joan
Frick Michele M.
Huang Hung Chang
Huang Timothy Yikai
Laroche Andre J.
Fox David T.
Greenlee Winner and Sullivan PC
Her Majesty the Queen in right of Canada as represented by the
Kruse David H
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