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In accordance with the present invention, there are provided transgenic plants comprising a plurality of plant-defense-associated proteins that are expressed to produce such proteins in an amount sufficient to increase the plants resistance to plant pathogens, relative to non-transgenic plants of the same species. The transgenic plants are useful to study patterns of development, and to provide increased resistance to plant pathogens when grown in crops as a food source, and the like. Nucleic acid constructs are also provided that are useful in methods for producing the invention transgenic plants.
1. A transgenic plant comprising:
a first exogenous transgene encoding an overexpressed and biologically active rice chitinase protein; and
a second exogenous transgene encoding an overexpressed and biologically active alfalfa glucanase protein,
said transgenes are constitutively expressed, and
said plant has increased resistance to fungal pathogens relative to plants that do not constitutively express both of said transgenes.
2. A plant according to claim 1, wherein said plant is a tobacco plant.
3. A nucleic acid construct comprising:
a first transgene encoding a biologically active rice chitinase protein; and
a second transgene encoding a biologically active alfalfa glucanase protein, and
wherein each of said transgenes are operatively linked to a plant-functional promoter.
4. A construct according to claim 3, wherein said promoter is a constitutive promoter.
5. A construct according to claim 3, wherein said construct imparts to transgenic plants containing same, increased resistance to fungal pathogens relative to non-transgenic plants of the same species that do not contain said construct.
6. A vector comprising the construct of claim 3.
7. A plant cell containing the vector of claim 6.
8. A method to increase fungal disease resistance of a plant, said method comprising:
introducing the nucleic acid construct of claim 3 into the genome of said plant.
9. A method to increase fungal disease resistance of a transgenic plant containing a transgene encoding a biologically active rice chitinase protein, said method comprising:
introducing a transgene encoding a biologically active alfalfa glucanase protein into the genome of said plant, wherein at least one transgene is heterologous to said plant.
10. A method to increase fungal disease resistance of a transgenic plant containing a transgene encoding a biologically active alfalfa glucanase protein, said method comprising:
introducing a transgene encoding a biologically active rice chitinase protein into the genome of said plant, wherein at least one transgene is heterologous to said plant.
The present invention relates to transgenic plants containing at least two plant-defense-associated transgenes.
The interactions between plants and various soil life forms are very complex, in some instances helpful to the plant and in other instances deleterious to the plant. Fungi harmful to plants (fungal pathogens) include fungal species from a wide variety of genera, including Fusarium, Pythium, Phytophthora, Verticillium, Rhizoctonia, Macrophomina, Thielaviopsis, Sclerotinia and numerous others. Plant diseases caused by fungi include pre- and post-emergence seedling damping-off, hypocotyl rots, root rots, crown rots, vascular wilts and a variety of other symptoms. Nematodes harmful to plants (nematode pathogens) include nematode species from the genera Meloidogyne, Heterodera, Ditylenchus, and Pratylenchus. Plant diseases caused by nematodes include root galls, root rot, lesions, "stubby" root, stunting, and various other rots and wilts associated with increased infection by pathogenic fungi. Some nematodes (e.g., Trichodorus, Lonoidorus, Xiphenema) can serve as vectors for virus diseases in a number of plants including Prunus, grape, tobacco and tomato.
Plant disease is the exception rather than the rule, as many plant pathogens express a virulent phenotype only on one or a limited number of host species. Pathogens inoculated onto a non-host species either lack the ability to grow and infect that plant, or following ingress invariably encounter a successful resistance mechanism. Interactions with host species exhibit a range of specificities dependent on the mechanism of the pathogen. One type of pathogen is the unspecialized "thug", which is necrotrophic and damages the host through production of toxins and/or enzymes. This pathogen is often equipped with inactivation or avoidance mechanisms to deal with host defenses. In contrast, "con men" pathogens grow biotrophically, avoiding serious host damage, at least in the early stages. Unlike thugs, con men pathogens do not activate host defenses nonspecifically.
For specialized pathogens having no avoidance mechanisms, host defenses are usually effective if induced. In specific interactions, following attempted infection by the pathogen, molecular signals determine whether the interaction will be incompatible or compatible. In an incompatible interaction (host resistant, pathogen avirulent), early molecular recognition is followed by rapid expression of defense responses. In a compatible interaction (host susceptible, pathogen virulent), the pathogen eludes the plant's surveillance mechanisms and disease generally ensues.
Various approaches have been utilized for attempting to control deleterious fungi and nematodes. One method is application of certain naturally occurring bacteria which inhibit or interfere with fungi or nematodes. See, for example, K. F. Baker and R. J. Cook, Biological Control of Plant Pathogens, Freeman and Co. (1974), for a description of fungi and nematodes and their interaction with plants, as well as a description of means for biological control of fungal and nematode pathogens. Another method is breeding for resistance, which is primarily focussed on the manipulation of minor resistance genes which make small quantitative contributions to the overall resistance of the plant.
Lytic enzymes have been individually transduced to form single-gene transgenic plants. For example, transfer of a tobacco basic vacuolar chitinase gene under the control of the CAMV35S promoter into the closely related species, N. sylvestris, did not give effective protection against C. nicotianae, even in transgenic plants exhibiting constitutively high levels of chitinase activity (Neuhaus et al., Plant Mol. Biol., 16:141-151, 1991). See also U.S. Pat. No. 4,940,840 to Suslow et al.
Recombinant bean chitinase has been expressed in transgenic tobacco seedlings (Broglie et al., Science, 254:1194-1197, 1991). The seedlings of the chitinase-containing transgenic plants have an enhanced, but not complete, resistance to a single species of fungi. Thus, it is desirable to obtain plants that have higher resistance levels to fungal pathogens than existing plants.
In accordance with the present invention, there are provided transgenic plants. Invention plants comprise a plurality of plant-defense-associated proteins that are expressed in an amount sufficient to increase the level of the plant's resistance to plant pathogens, relative to non-transgenic plants of the same species.
Plants or plant cells of the present invention are useful to study patterns of development, and to provide increased resistance to plant pathogens when grown in crops as a food source, and the like. Nucleic acid constructs are provided that are useful for producing the invention transgenic plants.
FIG. 1 shows the results of the Cercospora nicotinae fungal infection assay described in the Examples for the F4 generation invention transgenic tobacco plants. ∘=wild type tobacco plant, □=chitinase transgenic tobacco plant =glucanase transgenic tobacco plant, =chitinase/glucanase transgenic plant.
FIG. 2 shows the results of the Cercospora nicotinae fungal infection assay described in the Examples for the heterozygous F3 generation invention transgenic tobacco plants. □=wild type tobacco plant, ∘=chitinase transgenic tobacco plant, =glucanase transgenic tobacco plant, =chitinase/glucanase transgenic plant.
FIGS. 3A (day 4 data) and 3B (day 5 data) show the results of one of the Thanatephorus cucumeris fungal infection assays described in the Examples. The key in descending order corresponds to each cluster of bars from left to right on the bar graph. WT=wild type tobacco plant; GLC/3 and GLC/11=are glucanase transgenic tobacco plants; Cht/17=chitinase transgenic tobacco plant; X/7 and X/3 are chitinase/glucanase transgenic tobacco plants.
FIGS. 4A, 4B, and 4C show the results of one of the Thanatephorus cucumeris fungal infection assays described in the Examples. The key in descending order corresponds to each cluster of bars from left to right on the bar graph. WT =wild type tobacco plant; B2=glucanase transgenic tobacco plant; C2=chitinase transgenic tobacco plant; and C2B2=chitinase/glucanase transgenic tobacco plant.
In accordance with the present invention, there are provided transgenic plants comprising:
a plurality of transgenes wherein each transgene encodes a plant-defense-associated protein.
In another embodiment, there are provided transgenic plants comprising:
a first transgene encoding a first overexpressed plant-defense-associated protein; and
a second transgene encoding a second overexpressed plant-defense-associated protein, wherein the second plant-defense-associated protein is different from the first plant-defense-associated protein.
The term "plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and the like. Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Exemplary monocotyledons contemplated for use in the practice of the present invention include rice, wheat, maize, sorgham, barley, oat, forage grains, as well as other grains. Exemplary dicotyledons include tomato, tobacco, potato, bean, soybean, and the like.
The phrase "plants of the same species" refers to plants that have substantially completely identical genotypes, except for the inheritable nucleic acid transgenes that are introduced by the methods disclosed herein.
A "transgenic plant" refers to a plant or plant material that contains an inheritable expression cassette containing a recombinant transgene. A transgenic plant according to the present invention is a plant or plant material that contains one or more inheritable recombinant nucleic acid expression cassettes encoding at least 2 total plant-defense-associated proteins. Preferably the invention transgenic plant contains at least 3 plant-defense-associated proteins, more preferably at least 5, with at least 10 plant-defense-associated proteins being most desirable.
The plant-defense-associated proteins may be encoded by a gene that is foreign to the recipient plant (with respect to species to which the recipient belongs, i.e., heterologous/exogenous), foreign only to the particular individual recipient (i.e., exogenous), or genetic information already possessed by the recipient (i.e., endogenous).
When an expression cassette contains an endogenous gene encoding a naturally occurring plant-defense-associated protein, the cDNA for such endogenous gene is operatively linked to a promoter different from its native promoter, such that the gene can be overexpressed relative to expression levels that naturally occur in the non-invention transgenic plant, i.e., the plant is capable of producing higher levels of the encoded protein than are naturally produced.
As used herein, an "overexpressed" plant-defense-associated protein refers to a protein that is produced in higher amounts than are produced endogenously. Overexpression can be achieved, for example, by linking a transgene to an appropriate constitutive promoter, such that the transgene is continually expressed. Alternatively, the transgene can be linked to a strong, inducible promoter so that overexpression can occur on demand.
Suitable levels of overexpression include expression of the transgene about 1.5-fold up to about 1000-fold or more over the naturally occurring level of expression of the endogenous transgene. Preferred levels of overexpression are at least about 5-fold, with at least about 10-fold over the naturally occurring level of expression of the endogenous transgene being especially preferred.
In a preferred embodiment of the invention, the transgenic plant comprises a first exogenous transgene encoding a first heterologous plant-defense-associated protein; and a second exogenous transgene encoding a second heterologous plant-defense-associated protein.
A "heterologous protein" refers to a plant-defense-associated-protein encoded by a transgene obtained from a species different from the species into which the heterologous protein is transduced (i.e., a species different from the invention transgenic plant species). Protein (or polypeptide) is a term used herein to designate a linear series of amino acid residues connected one to the other by way of a peptide bond. The heterologous protein may have distinctive properties relative to the properties of endogenously produced antimicrobial defense proteins. The heterologous protein is preferably constitutively expressed, although the natural expression of the endogenous gene encoding the protein may be inducible in response to stress.
Suitable "plant-defense-associated-proteins" contemplated for use in the invention transgenic plants are those classes of proteins that are involved in specific plant defense mechanism pathways, such as, for example, lytic enzymes, thaumatine-like proteins, α-thionin (e.g., Bohlmann et al., EMBO J., 7:1559-1565, 1988), zeamatin (e.g., Vigers et al., Mol. Plant Micro. Interactions, 4:315-323, 1991) pathogenesis-related (PR) proteins (e.g., Bol et al., Ann. Rev. Phytopathol., 28:113-138, 1990), ribosome-inactivating-proteins (RIPs) (e.g., Leach et al., J. Biol. Chem., 266:1564-1573, 1990), lectins (e.g., Moreno et al., PNAS, USA, 86:7885-7889, 1989), cecropins, non-plant lysozymes, the Bacillus thuringensis toxin, enzymes involved in phytoalexin biosynthesis, proteinase inhibitors (e.g., Garcia-Olmedoz et al., Surv. Plant Mol. Cell Biol., 4:275-334, 1987), inducers of plant disease resistance mechanisms, and the like.
As used herein, "lytic enzyme" refers to a protein that is able to enzymatically cleave a bond or bonds of a molecule that is essential for the survival of a known pathogen. Exemplary lytic enzymes include chitinase, glucanase, cellulase, trehelase, and the like. See also, Boller T., (1987) "Hydrolytic enzymes in plant disease resistance." in T. Kosuge, E. W. Nester, eds. Plant-Microbe Interactions. Vol 2. Macmillan, New York, pp 385-413, incorporated herein by reference in its entirety.
As used herein, "chitinase" refers to an enzyme that is capable of degrading chitin. In one embodiment, a chitinase construct that constitutively expresses the chitinase protein is employed. Preferably, the chitinase gene employed herein is rice basic chitinase. An exemplary sequence encoding rice chitinase is set forth in SEQ ID NO:1.
Other suitable chitinase genes for use herein are described, for example, in U.S. Pat. No. 4,940,840 to Suslow, incorporated herein by reference. See also the list of published chitinase nucleotide sequences in Table 1 of Collinge et al., Plant Journal, 3:31-40, (1993), incorporated herein by reference. In addition, suitable chitinase genes can be obtained employing methods well-known in the art. For example, the nucleic acid molecule of SEQ ID NO:1 can be used as a probe to isolate related genes with chitinase activity from genomic or cDNA libraries of organisms known to produce chitinase proteins, such as Trichoderma harzianum, strain P1 (ATCC No. 74058; see also U.S. Pat. No. 5,173,419). See Sambrook et al., Molecular Cloning - - - A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y., (1989).
In another embodiment, a glucanase construct that constitutively expresses the endo-1,4-β-glucanase protein is employed. The term "glucanase" refers to an enzyme that is capable of degrading callose. Preferably, the glucanase gene employed herein is a class II alfalfa acidic β-glucanase. An exemplary sequence encoding a class II alfalfa β-glucanase is set forth in SEQ ID NO:3.
Other glucanase genes (cDNA) suitable for use herein are derived from: tomato (ATCC No. 68312; see also U.S. Pat. No. 5,168,064, incorporated herein by reference), avocado (Christoffersen et al, Plant Molec. Biol., 3:385, 1984) and bean (Tucker et al., Plant Physiol., 88:1257, 1988). In addition, see the tobacco glucanase sequences described in: Payne et al., Plant Mol. Biol., 15:797-808 (1990); Ward et al., Plant Physiol., 96:390-397 (1991); and the glucanase sequence described in Linthorst et al., PNAS,USA, 87:8756-8760 (1990). Suitable glucanase genes can also be obtained employing methods well-known in the art. For example, the nucleic acid molecule of SEQ ID NO:3 can be used as a probe to isolate related genes with glucanase activity (see Sambrook et al., supra).
In a presently preferred embodiment of the present invention, the first and second proteins employed in the transgenic plant are chitinase and glucanase, respectively.
The plant-defense-associated proteins described herein are encoded by recombinant transgene molecules. As used herein, the term "transgene" refers to a DNA or RNA molecule. Transgenes employed herein encode a biologically active amino acid sequence (i.e., a protein). The term "plurality of transgenes" refers to greater than or equal to 2.total transgenes encoding plant-defense-associated proteins. Preferably the invention transgenic plant contains at least 3 different transgenes encoding plant-defense-associated proteins, more preferably at least 5, with at least 10 different transgenes being most desirable. One of skill in the art will recognize that the transgenes employed herein encode the necessary signals required for expression of a biologically active protein, such as appropriate leader peptide sequences and the like. A biologically active protein is a protein that has at least one of the physiological properties exhibited under naturally occurring physiological conditions.
The transgenes encoding the plant-defense-associated proteins are typically contained in expression cassettes. The phrase "expression cassette" refers to a DNA molecule that is able to direct the transcription and translation of a structural gene (i.e., cDNA) so that a desired protein is synthesized. The expression cassette comprises at least one promoter operatively linked to at least one transgene encoding a desired protein, and a transcription terminator sequence. Thus, the protein-encoding segment is transcribed under regulation of the promoter region, into a transcript capable of providing, upon translation, the desired protein. Appropriate reading frame positioning and orientation of the various segments of the expression cassette are within the knowledge of persons of ordinary skill in the art; further details are given in the Examples.
The promoter region refers to the portion of a gene that controls transcription of DNA to which it is operatively linked. The promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. The particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of plant-defense-associated protein to increase the level of the plant's resistance to microbial infection. The amount of protein needed to induce resistance may vary with the type of plant. In a preferred embodiment, the promoter employed to express the transgenes is a constitutive promoter. It should be understood that this promoter may not be the optimal one for all embodiments of the present invention.
A "constitutive" promoter is a promoter which is active under all environmental conditions and all stages of development or cell differentiation. Constitutive promoters suitable for use in the practice of the present invention are widely available and are well known in the art. Exemplary constitutive promoters include the cauliflower mosaic virus 35S ("CaMV35S") promoter (see U.S. Pat. No. 5,097,925, incorporated herein by reference), CaMV19S promoter, nopaline synthase (NOS), octopine synthase (OCS), the rice actin gene promoter, and the like. In addition, the DNA promoter fragments from wheat described in U.S. Pat. No. 5,139,954; and plant promoters described in U.S. Pat. No. 5,097,025 are suitable for use herein. A presently preferred constitutive promoter for use in the practice of the present invention is CaMV35S.
The promoters used in the DNA constructs of the present invention may be modified, if desired, to affect their control characteristics. For purposes of this description, the phrase "CaMV35S" promoter thus includes variations of CaMV35S promoter, e.g., promoters derived by means of ligation with operator regions, random or controlled mutagenesis as well as, single, tandem or multiple copies of 35S enhancer elements, and the like.
In accordance with another embodiment of the present invention, there are provided nucleic acid construct(s) comprising the above-described expression cassette(s). The term "nucleic acid construct," or the abbreviated form "construct," as used herein, and throughout the specification and claims, refers to a recombinant nucleic acid molecule which can include expression cassettes, origins of DNA replication, procaryotic and eucaryotic genes from various sources (such as selectable marker genes), repressor genes, as well as any other sequence of nucleotides. The construct may be linear or in the circular form of a plasmid vector.
The nucleic acid construct of the present invention, including the segments of the expression cassette(s), are said to be "operably associated" with one another, such that said transgenes can be translationally expressed to produce the encoded protein under suitable conditions well-known to those of skill in the art.
As used herein the term "plasmid" or "vector" refers to circular, double-stranded DNA loops, which are not bound to the chromosome. One of skill in the art will recognize that the terms plasmid and vector can be used interchangeably. A plasmid contains DNA capable of causing expression of DNA sequences contained therein, where such sequences are in operational association with other sequences capable of effecting their expression, such as promoter sequences, and the like. The type and number of vectors employed is not critical, so long as greater than or equal to 2 transgenes are inheritable, e.g., capable of being expressed by each generation of plant. Suitable vectors for use in expressing the plant-defense-associated transgenes described herein include: pAMVBTS (ATCC No. 53637; Barton et al., Plant Physiol., 85:1103-1109 (1987); pBI101 (Jefferson et al., EMBO J., 6:3902-3907, 1987); and the pGEM and pSP vectors (Promega, Madison, Wis.). Presently preferred vectors for producing invention transgenic plants are the plasmids pBZ56, pM42X, and pBZ100, described hereinafter in the Examples section.
In accordance with yet another embodiment of the present invention, there are provided plant cells transformed with the above-described DNA construct(s).
The term "resistance," when used in the context of comparing the level of resistance between an invention transgenic plant and another plant, refers to the ability of the invention transgenic plant to maintain a desirable phenotype in the face of attack, relative to a non-transgenic plant or a single-gene transgenic plant. The level of resistance can be determined by comparing the physical characteristics of the invention plant to non-transgenic plants that either have or have not been exposed to microbial infection. Exemplary physical characteristics to observe include plant height, an increase in population of plants that have ability to survive microbial challenge (i.e., plants that come in contact with a compatible pathogen), delayed lesion development, reduced lesion size, and the like.
As used herein, "increased resistance to pathogens" refers to a level of resistance that an invention transgenic plant has to plant pathogens above a defined reference level. The defined reference level of resistance to a pathogen is the level of resistance displayed by non-transgenic or non-invention transgenic plants of the same species. Thus, the increased resistance is measured relative to previously existing plants of the same species. In one embodiment of the invention, the resistance is substantially increased above the defined reference level, e.g., greater than or equal to 20% above, preferably 50% above, more preferably 75% above; with up to 100% above being especially preferred.
The phrase "non-transgenic plants of the same species" means plants of the same species that do not contain any heterologous transgenes. The respective levels of pathogen resistance can be determined using well known methods including the Fungal Infection assays described hereinafter in the Examples section.
The phrase "transgenic plants of the same species that only express one exogenous transgene" refers to transgenic plants of the same species that only contain one transgene. These single-gene transgenic plants can contain any transgene including any one of the plurality of plant-defense-associated transgenes employed in the invention transgenic plant. Exemplary transgenic plants containing a single heterologous transgene include: glyphosate-resistant plants described in U.S. Pat. No. 5,188,642; and the plants described in Broglie et al., Science, 254:1194-1197 (1991); and Carmora et al., Plant J., 3:457-462 (1992).
In one embodiment of the present invention, the level of resistance imparted by the invention construct is "synergistic." Synergistic resistance refers to a level of resistance provided by at least two plant-defense-associated proteins in a single transgenic plant that is greater than the combined resistance observed for each protein contained individually in at least two single-gene transgenic plants..
Methods of introducing the constructs employed herein into suitable host cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art. According to the invention, the vector is introduced into the host cell by any suitable means, e.g., transformation employing plasmids, viral, or bacterial phage vectors, transfection, electroporation, lipofection, and the like. The heterologous DNA can optionally include sequences which allow for the extrachromosomal maintenance of the expression cassette, or said expression cassette construct can be caused to integrate into the genome of the host (as an alternative means to ensure stable maintenance in the host). See, for example: the Agrobacterium mediated transformation of germinating plant seeds described in U.S. Pat. No. 5,169,770; the plant potyvirus expression vector described in U.S. Pat. No. 5,162,601; U.S. Pat. No. 5,168,064, each of which are expressly incorporated herein by reference.
In addition, cauliflower mosaic virus (CaMV) may be used as a vector for introducing nucleic acid constructs into plant cells. (Hohn et al., "Molecular Biology of Plant Tumors," Academic Press, New York, pp . 549-560 (1982); Howell, U.S. Pat. No. 4,407,956). In accordance with the described method, the entire CaMV viral DNA genome is inserted into a parent bacterial plasmid creating a recombinant DNA molecule which can be propagated in bacteria. After cloning, the recombinant plasmid is further modified by introduction of the desired sequence into unique restriction sites in the viral portion of the plasmid. The modified viral portion of the recombinant plasmid is then excised from the parent bacterial plasmid, and used to inoculate the plant cells or plants.
Another suitable method of introducing DNA into plant cells is to infect a plant cell with Agrobacterium tumefaciens or A. rhizogenes that has previously been transformed with the gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefaciens) and hairy root disease (A. rhizogenes). The plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. In addition, assaying for the presence of opines can be used to identify transformed tissue.
Heterologous genetic sequences can be introduced into appropriate plant cells, for example, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-1183 (1987).
Ti and Ri plasmids contain two regions essential for the production of transformed cells. One of these, named transferred DNA (T-DNA), is transferred to plant nuclei and induces tumor or root formation. The other, termed the virulence (vir) region, is essential for the transfer of the T-DNA but is not itself transferred. The T-DNA will be transferred into a plant cell even if the vir region is on a different plasmid. Hoekema, et al., Nature, 303:179-189 (1983). The transferred DNA region can be increased in size by the insertion of heterologous DNA without affecting its ability to be transferred. A modified Ti or Ri plasmid, in which the disease-causing genes have been deleted, can be used as a vector for the transfer of the gene constructs of this invention into an appropriate plant cell.
Construction of recombinant Ti and Ri plasmids in general follows methods typically used with the more common bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to "shuttle vector," [Ruvkun and Ausubel, Nature, 298:85-88 (1981)], promoters, [Lawton et al., Plant Mol. Biol., 9:315-324 (1987)] and structural genes for antibiotic resistance as a selection factor [Fraley et al., Proc. Nat. Acad. Sci., 80:4803-4807 .(1983)].
There are two common ways to transform plant cells with Agrobacterium:
(1) co-cultivation of Agrobacterium with cultured isolated protoplasts (which requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts), or
(2) transformation of intact cells or tissues with Agrobacterium (which requires that the intact plant tissues, such as cotyledons, can be transformed by Agrobacterium, and that the transformed cells or tissues can be induced to regenerate into whole plants).
Most dicot species can be transformed by Agrobacterium. All species which are a natural plant host for Agrobacterium are transformable in vitro. Monocotyledonous plants, and in particular, cereals, are not natural hosts to Agrobacterium. Attempts to transform them using Agrobacterium have been unsuccessful until recently. Hooykas-Van Slogteren et al., Nature, 311:763-764 (1984). There is growing evidence now that certain monocots can be transformed by Agrobacterium. Indeed, cereal species such as rye (de la Pena et al., Nature, 325:274-275 (1987), corn (Rhodes et al., Science 240:204-207 (1988), and rice (Shimamoto et al., Nature, 338:274-276 (1989) may now be transformed.
In one embodiment of the present invention, two expression cassettes containing transgenes encoding chitinase and glucanase are prepared as described above. The expression cassettes are combined into a single expression vector, to form a DNA construct which comprises two individual genes encoding plant-defense-associated proteins. The vector is then inserted into cultured A. tumefaciens cells which contain a disarmed Ti plasmid. In another embodiment of the present invention, two vectors are employed containing one expression cassette each. The two vectors are either transformed into a single plant or into separate plants.
Two separate transgenic plants that each contain expression cassettes having at least one transgene can be sexually crossed using well-known methods to produce a transgenic plant of the present invention. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert the DNA constructs of this invention into plant cells. Such methods may involve, for example, the use of liposomes, electroporation, chemicals that increase free DNA uptake, and transformation using viruses. For example, the construct described above can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-185 (1985). The genetic material may also be transferred into the plant cell using polyethylene glycol, Krens, et al., Nature, 296:72-74 (1982).
Another method of introduction of transgene segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (see, for example, Klein, et al., Nature, 327:70-73, 1987). Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (see, for example, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79:1859-1863, 1982).
The DNA may also be introduced into the plant cells by electroporation. Fromm et al., Pro. Natl. Acad. Sci. USA, 82:5824 (1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
After transformation, transformed plant cells or plants comprising the invention nucleic acid constructs can be identified employing well-known methods. For example, a selectable marker, such as those discussed above, is typically used. Transformed plant cells can be selected by growing the cells on growth medium containing an appropriate antibiotic. The presence of opines can also be used if the plants are transformed with Agrobacterium.
After selecting the transformed cells, one can confirm expression of the desired heterologous gene. Simple detection of mRNA encoded by the inserted DNA can be achieved by well known methods in the art, such as Northern blot hybridization. The inserted sequence can be identified by Southern blot hybridization, as well.
Once the presence of the desired transgenes is confirmed, whole plant regeneration is desired. All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, Malus, Apium, Datura, and the like.
Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co. New York, 1983); and Vasil, I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III, 1986.
It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beet, cotton, fruit trees, and legumes.
Means for regeneration vary from species to species, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced employing well-known plant hormones in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
With the methods of the present invention, one can generate a transgenic plant containing at least a plurality of plant-defense-associated proteins, an origin of replication from either yeast, insect or mammalian cells, and a selectable marker gene for the expression of the plant-defense-associated proteins described herein.
The invention will now be described in greater detail by reference to the following non-limiting examples.
The nomenclature used hereafter and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning - - - A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y., (1989). The manual is hereinafter referred to as "Sambrook." Other general references are provided throughout this document. The procedures therein are well known in the art and are described herein for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Construction of plasmid pBZ56
A SphI fragment of rice chitinase RCH10 gene (SEQ ID NO: 1) from plasmid pRCH10 (described in Zhu et al., Plant J., 3:203-212, 1993) was subcloned into pSP72 (Promega, Madison, Wis.) to give pBZ5B. A Sac2/KpnI fragment of RCH10 was subcloned into pSP72 to give pBZ52. The NcoI/EcoRV fragment of pBZ52 was inserted into pBZ5B NcoI/PvuII sites to give pBZ54. The EcoRV/BgIII fragment of pBZ54 was inserted into pGEM721 BamHI/EcoRV sites to give pBZ55 [pGEM721 is a pGEM7 (Promega, Madison, Wis.) plasmid containing CaMV 35S promoter]. The HpaI/SacI fragment of pBZ55 was inserted into pBI101.1 (Jefferson et al., EMBO J., 6:3902-3907, 1987) to give pBZ56.
Construction of pM42X
A lambda ZAPII (Stratagene, La Jolla, Calif.) cDNA library prepared from poly (A) RNA isolated from alfalfa suspension cells 2, 3, and 4 h after elicitation with a fungal cell wall preparation (Dalkin et al., Physiol. and Mol. Plant Path., 37:293-307, 1990) was screened using a bean glucanase cDNA as probe (Edington et al., Plant Mol. Biol., 16:81-94, 1991). Positive plaques identified on duplicate filters were purified through two subsequent rounds of screening. Plasmids were then rescued in pBluescript SK- by use of the helper phage R408 (Stratagene), and insert size and diversity determined by restriction mapping. The largest insert that hybridized to the bean glucanase probe was subcloned into pGEM-3Zf(+) (Promega) to yield the plasmid "pAglu1", which contains the entire coding region for an isoform of acidic alfalfa β-1,3-glucanase (SEQ ID NO: 3).
A BamHI/XhoI fragment of pAglul containing an alfalfa β-glucanase gene was inserted into pMON530 (Monsanto, St. Louis, Mo.) Bg1II/XhoI sites to give pM4. The BamHI complete/PstI partial digested fragment of pM4 was inserted into pSP72 (Promega) to give pM42. The PvuII/SacI fragment of pM42 fragment was inserted into the SmaI/SacI site of pBI121 (Jefferson et al., EMBO J., 6:3902-3907, 1987) to give pM42X.
Construction of pBZ100
The plasmid pBZ55, described above, was digested with EcoRV/SalI, filled in with DNA polymerase Klenow fragment and religated to give pBZ55M. An HpaI/XhoI fragment of pBZ55M was inserted into pGEM7 (Promega) to give pBZ55M-7. A SacI fragment of pBZ55M-7 was inserted into pM42X to give pBZ100.
Plasmids pBZ56 and pM42X were directly transformed in Agrobacterium tumefaciens LBA4404, and independent transgenic tobacco plants containing either a rice chitinase or alfalfa glucanase transgene were generated by the leaf disc method (see, e.g., Rogers et al., Meth. in Enzymol., 118:627-640, 1986). Transformed plants were selected on Murashige and Skoog medium (see Murashige and Skoog, Physiol. Plant, 15:673, 1962) containing 200 μg/ml kanamycin and 500 μg/ml carbenicillin, and grown at 25� C. under a 16 hour light (115 mE)/8 hour dark cycle for several weeks. The seedlings from the transformed plants were then moved into soil.
Approximately 24 pBZ56 and 20 pM42X transgenic plants were confirmed as transformants by Southern blot hybridization. Expression of rice chitinase and alfalfa-glucanase transgenes was checked by conventional Northern blot and Western blot analysis. Several F2 generation transgenic lines that expressed high levels of either one of the above transgenes were selected for mating to produce an invention transgenic plant.
The F2 high level expression homozygous transgenic lines of pBZ56 and pM42X transformants were crossed with each other using routine methods. Polymerase chain reaction (PCR) analysis was employed to confirm that the heterozygous F3 generation (i.e., crossed plants) contained both transgenes. After confirmation, the F3 generation seeds were harvested and F4 generation plants were produced. F4 generation plants that were homozygous for both the chitinase and glucanase transgenes were selected and their genotypes were confirmed by back-cross and PCR analysis. The F4 plants were used for fungal infection assays.
The plasmid pBZ100 and a plasmid containing a hygromycin-resistant gene were introduced into rice embryos employing a commercially available ballistic micro-projectile device. The bombarded embryos were grown on N6 medium (see Chu et al., Scientia Sinica, 18:659-668, 1975) containing hygromycin. Regenerated hygromycin resistant plants were analyzed by PCR and Southern blot analysis for the presence of inheritable pBZ100 DNA. The results indicate that the genomic DNA of the rice transformants contain inheritable copies of both transgenes.
Both F3 and F4 generation invention transgenic tobacco plants, described above, were subjected to the following fungal infection assay. The results are shown in FIGS. 3 and 4, respectively.
Cercospora nicotianae (available from American Type Culture Collection under ATCC Nos. 18366 and 18367) were cultured by suspending mycelial fragments in sterile ddH2 O. A tobacco leaf suspension was prepared containing 200 ml V8 juice, 3.0 g CaCO3, 800 ml water, pH to 6.15, 18 g agar, 1 g dried tobacco leaves (dry several large leaves in the microwave for about 10 min or `defrost`; grind in a mortar and pestle), and autoclaved for 20-25 minutes. Approximately 0.5-1.0 ml of the tobacco leaf suspension was spread onto a petri plate. The contents of the plate were incubated at 25� C. with continuous light for about 4-6 weeks until white-ish mycelial covered the plate evenly.
The F3 and F4 generations of an invention tobacco transgenic plant (approximately 100 of each), and appropriate controls, were grown to about 5-6 weeks old. Mycelia were cut evenly into small squares (approximately 2 mm2) with a sterile scalpel. The mycelial agar squares were adhered to the underside and between the veins of tobacco leaves (not the newest leaf: usually the 2nd or 3rd from the top) using 3/4" waterproof surgical tape (Blenderm, 3M). The plants were incubated at 20� C., for 4-10 days using a 16 hour daylength.
Symptoms of fungal infection become visible in approximately 24-96 hours. Initially, small pin-pricks are just visible, with perhaps some browning of the leaf tissue over the inoculum. This progresses to a grey-ish, dry lesion of increasing diameter. The relative level of fungal infection was scored using a numerical rating system as follows:
0--no symptoms, maybe a little browning;
1--`pin-pricks,` but no contiguous, measurable lesion;
>1--(measuring the lesions, taking the widest portion as one measurement, then the perpendicular as the other; multiply these to get an approximate disease area in mm2)
FIG. 1 shows the results of the fungal infection assay for the F4 generation invention transgenic tobacco plants. The results indicate that the lesion sizes for the invention transgenic plant were at all times less than the lesion sizes observed for the controls, e.g., the wild type tobacco plant and the single-gene trangenic tobacco plants containing only one of either the chitinase or β-glucanase genes. Thus, the invention transgenic plant has a higher level of resistance to lesions caused by fungal pathogens than each of the control tobacco plants.
FIG. 2 shows the results of the fungal infection assay for the heterozygous F3 generation invention transgenic tobacco plants. The results indicate that the infection frequency for the invention transgenic tobacco plants was essentially less than the infection frequencies observed for the controls, e.g., the wild type tobacco plant and the single-gene trangenic tobacco plants containing only one of either the chitinase or β-glucanase genes. Thus, the invention transgenic plant has a higher level of resistance to fungal infection caused by fungal pathogens than each of the control tobacco plants.
In the imperfect stage, T. cucumeris from anastomosis group 3 (such as the strain ATCC No. 62149) causes stem lesions ("sore shin") similar to the pathogen Rhizoctonia solani. However, basidiospores of the fungus form in the perfect stage. Germinating basidiospores form appressoria that penetrate tobacco leaves directly, leading to the formation of lesions (`target spot`). Factors that favor development of the perfect stage are temperatures within the range of 16�-28� C. and leaf wetness.
T. cucumeris were cultured by inoculating autoclaved rice media (50 g Uncle Ben's Converted Rice, 25 ml doubly distilled water in 250 ml flasks; cover with foil and autoclave 25 minutes, see Shew and Main, Plant Disease, 74:1009-1013, 1990) with squares of mycelia from PDA (potato dextrose agar) plates. The plates were incubated at 22�-25� C. for 2-4 weeks with continuous light (the rice looks "mummified`- - - white and powdery).
To inoculate the invention transgenic tobacco plant, and appropriate controls, inoculum was ground in a blender until very few rice grains remained (approximately 3�45 seconds, scraping down the sides in between). Starting with the 0 level, inoculum was mixed into dry potting mix at the rate of 0, 1, 2 and 5 g rice mixture/liter of soil and distributed to 9-well pot forms. Eighteen 4-5 week old tobacco seedlings were transplanted to the wells, being careful to avoid carry-over from high inoculum levels to low ones (preferably use the wooden end of a separate disposable cotton applicator for each level to make a hole and firm the soil around the stem). Subsequently, pot-sets were placed in a flat cover with a plastic dome and incubated at 20� C. for 4-7 days using a 16 hour daylength. The dome was removed and sprinkled with water as needed to maintain moist conditions.
Symptoms of fungal infection in the highest inoculum become evident in approximately 3-4 days. Leaves touching the soil become grey-ish and wilted; upper leaves wilt; stems become girdled at the soil line, brown-ish and constricted. For the results shown in FIGS. 3A and B, the relative level of fungal infection was scored using a numerical rating system as follows:
0--No symptoms;
1--A diseased leaf or wilting, but plant may survive;
2--Severely constricted stem indicating plant will die; flat-out, wilted plant.
The results of the assay at days 4 and 5 are shown in FIGS. 3A and 3B, respectively. FIG. 3A (day 4) indicates that the invention transgenic plant designated X/3, when grown in potting soil containing 5 g of the above-described fungal rice mixture/liter, has a higher level of resistance to the pathogen than the control tobacco plants which include a wild type tobacco plant and single-gene trangenic tobacco plants containing either one of the chitinase or β-glucanase gene.
FIG. 3B (day 5) indicates that both the X/7 and X/3 invention transgenic tobacco plants, when grown in potting soil containing 2 g and 5 g of rice mixture/liter, have a higher level of resistance to the pathogen than the control tobacco plants which include a wild type tobacco plant and single-gene trangenic tobacco plants containing either one of the chitinase or β-glucanase gene.
A similar assay was conducted using F5 transgenic tobacco plants that were homozygous for both the chitinase and β-glucanase genes. In this assay 4 g, 15 g, and 45 g of the rice mixture/liter was employed, and the plants were scored as either healthy or diseased. The results are shown in FIGS. 4A (4 g), 4B (15 g), and 4C (45 g). The results indicate that the invention transgenic plant containing two plant-defense-associated proteins clearly has increased resistance to fungal pathogens relative to non-transgenic plants and transgenic plants of the same species that only express one of said plant-defense-associated proteins.
While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1151 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: both(D) TOPOLOGY: both(ii) MOLECULE TYPE: cDNA(ix) FEATURE: (A) NAME/KEY: CDS(B) LOCATION: 55..1062(D) OTHER INFORMATION: /product="RICE CHITINASE"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AATCAGTCAATCTGTATACAGCAACTCAGCGATCTTATATTTACCCAACACACCATG57 MetAGAGCGCTCGCTGTGGTGGCCATGGTGGCCAGGCCCTTCCTCGCGGCG105ArgAlaLeuAlaValValAlaMetValAlaAr gProPheLeuAlaAla51015GCCGTGCATGCCGAGCAGTGCGGCAGCCAGGCCGGCGGCGCGGTGTGC153AlaValHisAlaGluGlnCysGlySerGlnAl aGlyGlyAlaValCys202530CCCAACTGCCTCTGCTGCAGCCAGTTCGGCTGGTGCGGCTCCACCTCC201ProAsnCysLeuCysCysSerGlnPheGlyTrpCy sGlySerThrSer354045GACTACTGCGGCGCCGGATGCCAGAGCCAGTGCTCGCGGCTGCGGCGG249AspTyrCysGlyAlaGlyCysGlnSerGlnCysSerArgLe uArgArg50556065CGGCGGCCCGACGCGTCCGGCGGCGGTGGCAGCGGCGTCGCGTCCATC297ArgArgProAspAlaSerGlyGlyGlyGlySerGl yValAlaSerIle707580GTGTCGCGCTCGCTCTTCGACCTGATGCTGCTCCACCGCAACGATGCG345ValSerArgSerLeuPheAspLeuMetLeuLe uHisArgAsnAspAla859095GCGTGCCCGGCCAGCAACTTCTACACCTACGACGCCTTCGTCGCCGCC393AlaCysProAlaSerAsnPheTyrThrTyrAs pAlaPheValAlaAla100105110GCCAGCGCCTTCCCGGGCTTCGCCGCCGCGGGCGACGCCGACACCAAC441AlaSerAlaPheProGlyPheAlaAlaAlaGlyAs pAlaAspThrAsn115120125AAGCGCGAGGTCGCCGCGTTCCTTGCGCAGACGTCCCACGAGACCACC489LysArgGluValAlaAlaPheLeuAlaGlnThrSerHisGl uThrThr130135140145GGCGGGTGGGCGACGGCGCCCGACGGCCCCTACACGTGGGGCTACTGC537GlyGlyTrpAlaThrAlaProAspGlyProTyrTh rTrpGlyTyrCys150155160TTCAAGGAGGAGAACGGCGGCGCCGGGCCGGACTACTGCCAGCAGAGC585PheLysGluGluAsnGlyGlyAlaGlyProAs pTyrCysGlnGlnSer165170175GCGCAGTGGCCGTGCGCCGCCGGCAAGAAGTACTACGGCCGGGGTCCC633AlaGlnTrpProCysAlaAlaGlyLysLysTy rTyrGlyArgGlyPro180185190ATCCAGCTCTCCTACAACTTCAACTACGGGCCGGCGGGGCAGGCCATC681IleGlnLeuSerTyrAsnPheAsnTyrGlyProAl aGlyGlnAlaIle195200205GGCGCCGACCTGCTCGGCGACCCGGACCTCGTGGCGTCTGACGCCACC729GlyAlaAspLeuLeuGlyAspProAspLeuValAlaSerAs pAlaThr210215220225GTCTCCTTCGACACGGCCTTCTGGTTCTGGATGACGCCGCAGTCGCCC777ValSerPheAspThrAlaPheTrpPheTrpMetTh rProGlnSerPro230235240AAGCCGTCGTGCAACGCGGTCGCCACCGGCCAGTGGACGCCCTCCGCC825LysProSerCysAsnAlaValAlaThrGlyGl nTrpThrProSerAla245250255GACGACCAGCGGGCGGGCCGCGTGCCGGGCTACGGCGTCATCACCAAC873AspAspGlnArgAlaGlyArgValProGlyTy rGlyValIleThrAsn260265270ATCATCAACGGCGGGCTGGAGTGCGGCCATGGCGAGGACGATCGCATC921IleIleAsnGlyGlyLeuGluCysGlyHisGlyGl uAspAspArgIle275280285GCCGACCGGATCGGCTTCTACAAGCGCTACTGCGACATCCTCGGCGTC969AlaAspArgIleGlyPheTyrLysArgTyrCysAspIleLe uGlyVal290295300305AGCTACGGCGCCAACTTGGATTGCTACAGCCAGAGGCCTTCGGCTCCT1017SerTyrGlyAlaAsnLeuAspCysTyrSerGlnAr gProSerAlaPro310315320CCTAAGCTTCGCCTACCTAGCTTCCACACAGTGATAAATAATCAC1062ProLysLeuArgLeuProSerPheHisThrVa lIleAsnAsnHis325330335TGATGGAGTATAGTTTACACCATATCGATGAATAAAACTTGATCCGAATTCTCGCCCTAT1122AGTGAGTCGTATTAGTCGACAGCTCTAGA 1151(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 336 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetArgAlaLeuAlaValValAlaMetValAlaArgProP heLeuAla151015AlaAlaValHisAlaGluGlnCysGlySerGlnAlaGlyGlyAlaVal202530 CysProAsnCysLeuCysCysSerGlnPheGlyTrpCysGlySerThr354045SerAspTyrCysGlyAlaGlyCysGlnSerGlnCysSerArgLeuArg50 5560ArgArgArgProAspAlaSerGlyGlyGlyGlySerGlyValAlaSer65707580IleValSerArgSerLeuPhe AspLeuMetLeuLeuHisArgAsnAsp859095AlaAlaCysProAlaSerAsnPheTyrThrTyrAspAlaPheValAla1001 05110AlaAlaSerAlaPheProGlyPheAlaAlaAlaGlyAspAlaAspThr115120125AsnLysArgGluValAlaAlaPheLeuAlaGlnThrSerH isGluThr130135140ThrGlyGlyTrpAlaThrAlaProAspGlyProTyrThrTrpGlyTyr145150155160Cys PheLysGluGluAsnGlyGlyAlaGlyProAspTyrCysGlnGln165170175SerAlaGlnTrpProCysAlaAlaGlyLysLysTyrTyrGlyArgGly 180185190ProIleGlnLeuSerTyrAsnPheAsnTyrGlyProAlaGlyGlnAla195200205IleGlyAlaAspLeuLeuGly AspProAspLeuValAlaSerAspAla210215220ThrValSerPheAspThrAlaPheTrpPheTrpMetThrProGlnSer225230235 240ProLysProSerCysAsnAlaValAlaThrGlyGlnTrpThrProSer245250255AlaAspAspGlnArgAlaGlyArgValProGlyTyrG lyValIleThr260265270AsnIleIleAsnGlyGlyLeuGluCysGlyHisGlyGluAspAspArg275280285Ile AlaAspArgIleGlyPheTyrLysArgTyrCysAspIleLeuGly290295300ValSerTyrGlyAlaAsnLeuAspCysTyrSerGlnArgProSerAla305 310315320ProProLysLeuArgLeuProSerPheHisThrValIleAsnAsnHis325330335(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1374 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: both(D) TOPOLOGY: both(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 52..1158(D) OTHER INFORMATION: /product="ALFALFA BETA-GLUCANASE"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:CGGCAAATCCTTCTTTC ATATTCATTTTTAGTGTATACTTTATTTTGCATCATGCCT57MetPro1TCTTTCTTT GCTCCAACCAGGAGGTTCTCCTTGGCTTCTCCTCTCCTT105SerPhePheAlaProThrArgArgPheSerLeuAlaSerProLeuLeu51015CTATTGGGATTG TTCACAATAAACCTCATTCCCACAGCAGATGCTCAA153LeuLeuGlyLeuPheThrIleAsnLeuIleProThrAlaAspAlaGln202530ATAGGAGTATGTTATGGT ATGATGGGAAACAATCTACCACCAGCAAAC201IleGlyValCysTyrGlyMetMetGlyAsnAsnLeuProProAlaAsn35404550GAAGTTATAGAT CTCTACAAAGCAAACAACATTAAGAGAATGAGACTC249GluValIleAspLeuTyrLysAlaAsnAsnIleLysArgMetArgLeu556065TATGATCCT AATCAAGCTGCTCTAAATGCATTAAGAAATTCAGGCATT297TyrAspProAsnGlnAlaAlaLeuAsnAlaLeuArgAsnSerGlyIle707580GAACTCATT CTTGGTGTGCCTAATTCCGACCTTCAAAGCCTAGCCACC345GluLeuIleLeuGlyValProAsnSerAspLeuGlnSerLeuAlaThr859095AACTCTGATAAT GCACGTCAATGGGTACAAAGAAATGTATTGAATTTC393AsnSerAspAsnAlaArgGlnTrpValGlnArgAsnValLeuAsnPhe100105110TGGCCTAGTGTCAAAATC AAGTATATTGCAGTTGGTAATGAAGTGAGT441TrpProSerValLysIleLysTyrIleAlaValGlyAsnGluValSer115120125130CCAGTTGGAGGT TCTTCTTGGCTAGGACAATATGTTTTACCTGCCACC489ProValGlyGlySerSerTrpLeuGlyGlnTyrValLeuProAlaThr135140145CAAAATATA TATCAAGCTATAAGAGCTAAAAATCTTCATGATCAAATC537GlnAsnIleTyrGlnAlaIleArgAlaLysAsnLeuHisAspGlnIle150155160TTGGTTTCA ACCGCTATTGACATGACCCTTATTGGAAACTCATTCCCT585LeuValSerThrAlaIleAspMetThrLeuIleGlyAsnSerPhePro165170175CCATCTAAAGGT TCTTTCAGAAATGATGTTAGGGCATACCTAGATCCT633ProSerLysGlySerPheArgAsnAspValArgAlaTyrLeuAspPro180185190TTTATTGGATACTTGGTA TATGCAGGTGCACCTTTACTTGTCAATGTT681PheIleGlyTyrLeuValTyrAlaGlyAlaProLeuLeuValAsnVal195200205210TACCCTTATTTT AGCCATGTTGGTAACCCGCGCGACATATCTCTTCCT729TyrProTyrPheSerHisValGlyAsnProArgAspIleSerLeuPro215220225TATGCTCTT TTCACTTCACCGGGTGTTATGGTACAAGACGGTCCAAAT777TyrAlaLeuPheThrSerProGlyValMetValGlnAspGlyProAsn230235240GGGTACCAA AACTTGTTTGATGCTATGTTGGATTCGGTGCATGCAGCC825GlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHisAlaAla245250255CTAGATAACACT GGGATTGGTTGGGTGAACGTTGTTGTATCTGAGAGT873LeuAspAsnThrGlyIleGlyTrpValAsnValValValSerGluSer260265270GGTTGGCCCTCTGATGGA GGCGCTACTTCATATGACAACGCACGTATT921GlyTrpProSerAspGlyGlyAlaThrSerTyrAspAsnAlaArgIle275280285290TATCTTGATAAT TTGATTCGTTATGAAGGTAAAGGTACTCCAAGAAGG969TyrLeuAspAsnLeuIleArgTyrGluGlyLysGlyThrProArgArg295300305CCTTGGGCT ACAGAAACTTATATTTTTGCTATGTTTGATGAGAACCAA1017ProTrpAlaThrGluThrTyrIlePheAlaMetPheAspGluAsnGln310315320AAGAGTCCA GAATTGGAGAAACATTTTGGAGTGTTTTATCCTAATAAA1065LysSerProGluLeuGluLysHisPheGlyValPheTyrProAsnLys325330335CAAAAGAAGTAC CCATTTGGATTTGGTGGGGAAAGAATGGGAATTGTC1113GlnLysLysTyrProPheGlyPheGlyGlyGluArgMetGlyIleVal340345350AATGGTGACTTCAATGCA ACTATTTCTCTTAAGAGTGACATGTAAGAAAAAA1165AsnGlyAspPheAsnAlaThrIleSerLeuLysSerAspMet355360365ATCAAGGTTTTCAAGATTTGAGTGGTTTTATGCATAAAAT AAGAGAATTTCTCGTGTATG1225TATTTTATTTATCTTTCTTTTTTCGGGTGTAGAAAATTTGGAATGCTTGAGTTTCCTATT1285TCTCTAAAAATTAATGTCTTGTGATGAAGTTATATGAATATTTATATAAAGCGTAACTTT1345CCAAATTATTTAATA CTATTTTTCAAAAA1374(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 368 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:MetProSerPhePheAla ProThrArgArgPheSerLeuAlaSerPro151015LeuLeuLeuLeuGlyLeuPheThrIleAsnLeuIleProThrAlaAsp20 2530AlaGlnIleGlyValCysTyrGlyMetMetGlyAsnAsnLeuProPro354045AlaAsnGluValIleAspLeuTyrLysAlaAsnAsnI leLysArgMet505560ArgLeuTyrAspProAsnGlnAlaAlaLeuAsnAlaLeuArgAsnSer65707580 GlyIleGluLeuIleLeuGlyValProAsnSerAspLeuGlnSerLeu859095AlaThrAsnSerAspAsnAlaArgGlnTrpValGlnArgAsnValLeu 100105110AsnPheTrpProSerValLysIleLysTyrIleAlaValGlyAsnGlu115120125ValSerProValGlyGly SerSerTrpLeuGlyGlnTyrValLeuPro130135140AlaThrGlnAsnIleTyrGlnAlaIleArgAlaLysAsnLeuHisAsp1451501 55160GlnIleLeuValSerThrAlaIleAspMetThrLeuIleGlyAsnSer165170175PheProProSerLysGlySerPheArgAsnAspV alArgAlaTyrLeu180185190AspProPheIleGlyTyrLeuValTyrAlaGlyAlaProLeuLeuVal195200205 AsnValTyrProTyrPheSerHisValGlyAsnProArgAspIleSer210215220LeuProTyrAlaLeuPheThrSerProGlyValMetValGlnAspGly225 230235240ProAsnGlyTyrGlnAsnLeuPheAspAlaMetLeuAspSerValHis245250255AlaAlaLeuAspAsn ThrGlyIleGlyTrpValAsnValValValSer260265270GluSerGlyTrpProSerAspGlyGlyAlaThrSerTyrAspAsnAla2752 80285ArgIleTyrLeuAspAsnLeuIleArgTyrGluGlyLysGlyThrPro290295300ArgArgProTrpAlaThrGluThrTyrIlePheAlaMetPheA spGlu305310315320AsnGlnLysSerProGluLeuGluLysHisPheGlyValPheTyrPro32533033 5AsnLysGlnLysLysTyrProPheGlyPheGlyGlyGluArgMetGly340345350IleValAsnGlyAspPheAsnAlaThrIleSerLeuLysSerAspMet 355360365__________________________________________________________________________
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By:Kyle Caso
Saturday February 19th the Brooklyn Nets and Sacramento Kings agreed on a deal that would send 6"4, 26 year old shooting guard Marcus Thornton to brooklyn for 36 year old shooting guard jason Terry and 33 year old power forward Reggie Evans.
This is the second trade the Nets made this season besides the blockbuster deal for Paul Pierce and Kevin Garnett. First the Nets sent Tyshawn Taylor and Tornike Shengela to the Bulls for Marquise Teague. Now the nets have sent Jason Terry and Reggie Evans to the Kings for Marcus Thornton.
Marcus Thornton is 26 years of age and is a scoring SG. He has had an up and down season for the Kings although his production has been parallel to his role in the offense. Marcus has proven he can score at will. When Marcus plays 30.0 Minuets or more he averages 20.4 points per game. Marcus even dropped 42 points on the number one seed in the east.
Jason Terry Is 36 and has missed 16 games this year due to injury and even when he is on the court has not produced much at all outside of an 18 point performance in which the Nets lost to Detroit. Reggie Evans is a 33 year old rebounding Power forward who has proved he can produce as far as rebounding. Reggie's offense on the other hand is just below the average of the below average mark.
Overall the trade worked well for both teams. The Nets walked away with the better player(s)
and the Kings walked away with salary relief. Marcus Thornton is due 17 million the rest of the season and the end of next season. This doesn't really concern the Nets seeing as that there big focus is on the free agency of 2016.

subset: commoncrawl


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Golf & Business
Can "The Squeeze" buck the trend of bad golf movies?
Terry Jastrow is the writer and director of "The Squeeze," a new golf movie opening in theaters and video on demand Friday. Jastrow, 66, worked for more than 20 years as a producer and...
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Tenuous golf connection
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Remembering Harold Ramis, the non-golfing golf hero
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Monday, 2/24/2014 at 3:23PM
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- Harold Ramis,
- caddyshack,
- funny,
- golf movies
Gear & Equipment
Five questions with 'The Short Game' director Josh Greenbaum
Josh Greenbaum directed "The Short Game," a documentary that tracks eight 7-year-olds at the 2012 U.S. Kids Golf World Championship. The film debuts today in select cities. He answers five questions from Mike Stachura....
Friday, 9/20/2013 at 9:34AM
- keywords:
- golf,
- golf movies,
- josh greenbaum,
- mike stachura,
- the short game
Magazines / Editors' Blog
More readers weigh in on the sorry state of golf cinema.
Golf World's recent Arts Issue covered the generally lame state of golf movie-making, pointing out that most of the successful and palatable movies were comedies ("Happy Gilmore," "Tin Cup," "Caddyshack"). Our reader mail made a...
Tuesday, 9/27/2011 at 3:49PM
- keywords:
- Caddyshack,
- Dead Solid Perfect,
- Golf World,
- Golf World Arts Issue,
- Happy Gilmore
Magazines / Editors' Blog
Hey, Golf World, you missed a good golf movie
Golf World's Arts Issue, out this week, has already drawn comment about its coverage of golf fiction and movies, including this letter reminding the editors of a movie that they might have missed in compiling...
Friday, 9/23/2011 at 9:53AM
- keywords:
- A Gentleman's Game,
- Arts,
- Brian Doyle-Murray,
- Follow the Sun,
- Gary Sinise
Magazines / Editors' Blog
More suggestions for Golf World's Arts Issue
We continue to receive mail about Golf World's November 22 Arts Issue, which covered books, movies and painting, most with suggestions for other artists we might have included. Our old friend Tom Stewart, who runs...
Sunday, 12/5/2010 at 8:58PM
- keywords:
- Bill Fields,
- Billy Mac,
- Golf Worlds,
- Old Sport Gallery,
- Richard Chorley
Golf Tours & News / Opinion
Bill Fields: Golf And Film Can Be A Challenge To Pair
Why golf movies are rarely as good as the book
November 22, 2010
- keywords:
- golf movies,
- golf world,
- caddyshack,
- Bill Fields,
- tin cup
Results: 1 - 8 of 8


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Tariffs: the bottom line, #1 contributing factor, is low cost of labor due to lack of labor laws overseas. The U.S. and other nations have all kinds of laws and regulations to protect the laborer, from OSCA to minority protection against discrimination. All that adds to the cost of having employees. Quite simply, China and others don't have 90% of those laws and regulations. Until that disparity is corrected, there will never be a level playing field and U.S. manufacturing will be at an insurmountable disadvantage.
Tariffs can be one answer. Presently, the U.S. doesn't want to tariff China because of foreign relations etc. There may be one possible excuse which could get by: linking a tariff to the disparity of labor protection laws. If China protests,world opinion will crucify them.


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yes, bell hooks, the quintessential feminist / philosopher / educator herself will be in-store Monday November 19th at 5:30 pm promoting and signing her latest collection of poetry, Appalachian Elegy: Poetry & Place; 80 pages and 66 poems of her poetic insights and personal aesthetics shaped by Appalachian birthright and worldly experiences as an educator and cultural critic. The Wild Fig Books is deeply honored to be the chosen stage for such a highly admired and beloved American Icon.
Unable to attend? Then perhaps you'd like to pre-order a signed copy now to be mailed directly to you or elsewhere as a gift to a friend, teacher, or other inspirational loved-one, acquaintance or influence. Book price is $19.95 plus tax and $2.99 for domestic shipping fees. Please contact us as early as possible to reserve your copy by any of the following means:
via email: email@example.com
via social network: facebook.com/thewildfigbooks
via in-store phone: 859-381-8133
– thank you. hope to see you soon.


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VIDEOS: Empire Interviews with Jennifer and Josh
Empire got their chance to meet Katniss and Peeta, actors Jennifer Lawrence and Josh Hutcherson.
One of them is lethal with the bow and arrow; the other can hurl heavy objects long distances at death-inducing speeds. They're Katniss Everdeen and Peeta Mellark, heroes of Panem's Hunger Games, and pretty soon they'll be on poster walls across the globe. The stars who play them in The Hunger Games, Oscar-nominee Jennifer Lawrence and fellow Kentuckian Josh Hutcherson, recently downed weapons to meet their public. Empire, who, to paraphrase Blackadder, can usually muster all the killing power of an asthmatic ant with heavy shopping, jogged down to meet them and ask about scoring the coveted roles and getting all buffed up to enter the arena.
To play the interviews, click the Pictures below:
To play Josh's interview, click his link at the bottom of this page.
sourece: Empire


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hopefully its beast
0 comments by Admiral_Skeybar on May 31st, 2010
It's been a bit quiet on the production latetely but I'm happy to announce that it is currently going fine again.
The programmer was selling his house and this caused the delay, he has sold his house now and has time to work on Battle of the Coral Sea again.
A rough version of the game is slowly taking shape and more of the controls are functional. The sounds from the sound designer have been implemented and now we only need the music and the sound-part will be finished.
We are also making a website and we have already set up a FORUM.
Thank you for supporting us.
hopefully its beast
We are still busy with the source code and graphics engine so it will take a while until it's released but most models are finished already. :)
thats dope, im watching ever since i posted first comment =)
Yeh that's great, thanks :)
Promote Battle of the Coral Sea on your homepage or blog by selecting a button and using the embed code provided (more).


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7 Of The Worst Dressed Celebs In Hollywood
What Does Kylie Jenner Look Like Without Makeup?
Celebrity Offspring Who Are Rising to Fame
Is Meri Brown Quitting Sister Wives and Leaving Kody Brown?
Did You know These Stars Were Born Rich?
The Adorable Children of Reality TV Stars
8 Bra Hacks to Try for Yourself
Vivienne and Knox Jolie-Pitt Are All Grown Up
Kendall and Kylie Jenner Celebrate High School Graduation with Star-Studded Party
This 9-Year-Old Model Is Being Called the "Most Beautiful Girl in the World"
You Won't Believe How Brad and Angie Travel
Did JLo Break Up America's Sweethearts?
This Girl's Fake Pregnancy Prank Totally Backfired
These 9 Celeb Kids Dress Better Than You Do
Surprising Ways That Chia Seeds Can Change Your Life
WTFashion: Celebrities' Swimwear Style
Scott Disick Talks Baby Number 4 With Kourtney Kardashian
12 Fascinating Transgender Celebrities
Ashley Olsen's Bad Attitude Caused a Major Scene
Miley Cyrus and Her New Man Caught in The Act
Michael Jackson's Daughter Paris Reveals Her Boyfriend
Kristen Bell Leaves "Frozen" Voicemail For Ailing Fan That Will Totally Melt Your Heart
Celeb Kids Who Look Just Like Dad
Is Katy Perry America's Next Top Supermodel
Khloe Kardashian and Lamar Odom Make Divorce Official
How Is Jill Zarin Coping After Horrible Cancer News?
Best Celebrity Beach Bodies of 2015
Seeing Double: Celebrities Who Could Be Twins
Meet The Newest Member of The Duggar Clan


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About 19 percent of airborne fallout from the Fukushima nuclear disaster was deposited in Japan, and only about 2 percent made it to other land areas in Asia and North America, according to a study published this week by the European Geosciences Union. The bulk was absorbed by the Pacific Ocean.
Opponents of nuclear energy have seized on the European study because it describes the total release at Fukushima as "massive." It finds that twice as much cesium 137 was released at Fukushima than originally reported.
And it says "high concentrations" reached North America and Europe.
It examines two radionuclides in particular–xenon 133 and cesium 137–and measures the total release in terabecquerels.
"Altogether, we estimate that 6.4 TBq of 137Cs, or 19 percent of the total fallout until 20 April, were deposited over Japanese land areas, while most of the rest fell over the North Pacific Ocean," the authors write. "Only 0.7 TBq, or 2 percent of the total fallout were deposited on land areas other than Japan."
Scientists generally regard ocean absorption of fallout preferable to land deposits, because water absorbs radiation and the volume of the ocean dilutes it. On land, radioactive isotopes can be consumed by livestock and concentrated in milk and other food sources.
The study also notes that airborne emissions of cesium 137 continued until March 19, when Japanese authorities began spraying water on the spent fuel pool of reactor #4.
"This indicates that emissions were not only coming from the damaged reactor cores, but also from the spent-fuel pool of unit 4 and confirms that the spraying was an effective countermeasure."
Related Posts:
Harm From Fukushima Radiation: A Matter of Perspective
Four Sites Where You Can Monitor US Radiation Levels
Scientists Will Track Fukushima Radiation To Study Ocean Currents


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Kids Earn Bikes For Books
Some Caribou youngsters won the school lottery today, in a manner of speaking. As Shawn Cunningham reports, their hard work this school year has paid off in a BIG WAY that left some kids with some SNAZZY NEW WHEELS.
This program is made possible by the Caribou masonic Lodge.
Category: News


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Posted: November 8, 2011
Having ended a three-year hiatus just over a month ago, the Raconteurs seem to be in fighting form after closing out the Voodoo Experience in New Orleans on Oct. 30.
Here's a great video of the band playing their 2006 hit single, "Steady As She Goes," which is off Broken Boy Soldiers.
Check out guitarist – and former White Stripes front man and Dead Weather drummer – Jack White putting his customized Gretsch Duo Jet through the paces:
Tags: gretsch, Jack White, The Raconteurs, Voodoo Festival
Posted In: Artists Blogs | Leave a comment
Keep in touch with the latest Gretsch news, pictures and videos.
New From The Gretsch Blog.
What are you interested in?
View Past Blogs.


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Bud Norris now in leadership role with youthful Astros
Former Hooks ace now veteran on big-league club
CORPUS CHRISTI - With 97 starts in three-plus seasons, right-hander Bud Norris hardly can be classified as a salted major league veteran.
Yet the changes and experiences through which the former Hooks pitcher have undergone now place him in a unique position for a 27-year-old major leaguer — a veteran and a leader on a ballclub teeming with younger players.
"I should write a book," Norris quipped Thursday during the Astros' annual winter caravan stop.
Norris was part of the organization's contingent that included Astros mainstay Larry Dierker, former broadcaster Milo Hamilton, Astros and ex-Hooks pitcher Jordan Lyles, outfielder Justin Maxwell, Hooks manager Keith Bodie and Astros president and CEO George Postolos. The group also made visits at Gregory-Portland High School and the Christus Spohn Hospital Cancer Center and conducted an autograph session at Academy Sports & Outdoors.
Norris could be considered the Astros' elder statesman of sorts as the franchise moves to the American League West this season on the heels of a franchise-worst 55-107 record.
Of the 21 pitchers on Houston's active roster, no one has Norris' major league tenure and only two — relievers Fernando Rodriguez and Jose Veras — have pitched in more games. Of the 18 position players, only one, eight-plus-year journeyman infielder Carlos Pena, has more service time.
Thus, Norris has some additional duties as well as honing his pitches and staying healthy as he begins his fourth full season in the bigs.
"I've really had a different outlook on this upcoming season as far as where I need to be as far as a leader and one of the anchors of the staff. It's a new role. I only have three and a half years. I'm not a 10-year (guy) or a salty vet by any means," said Norris, who last Friday avoided arbitration by signing a one-year, $3-million contract, making him the highest-paid player on the team.
"But with my time here in Houston I can hopefully bring some things down to Jordan and some of the other younger pitchers and bring some things together," Norris said. "What really excites me is we're all close in age. We're all going through a lot of these life lessons together. That bonding association that we're going to have with each that's going to get bigger and bigger every day and you can really build from within."
That is the new plan in Houston with the arrival of new owner Jim Crane, general manager Jeff Luhnow, Postolos and new manager Bo Porter. New blood is being injected into the organization, whether by trade or a re-emphasis at the developmental level.
Change has been the catch phrase and Norris has seen plenty of it since his debut on July 29, 2009, a three-inning relief appearance against the Cubs in Chicago. Since then, there have been three pitching coaches — Dewey Robinson, Brad Arnsberg and, currently, Doug Brocail. The managerial list includes Cecil Cooper, former Hooks manager Dave Clark on an interim basis, Brad Mills and interim Tony DeFrancesco before Porter was hired in September.
That doesn't count the change in owners and front-office staff headed by new GM Luhnow, who helped craft two World Series champions in St. Louis.
"Obviously, I think that's the message that Luhnow is trying to portray to us that, hey, you go out and get a bunch of core players a lot can happen and you can really build something," Norris said.
That means the few veterans remaining on the Houston roster will be counted upon not only for production but for guidance as the Astros wean their new talent into the major league level.
Norris was 7-13 with a 4.65 earned-run average in 29 starts last season while battling blister, hip-flexor and shoulder ailments. In his three-plus seasons in Houston, Norris is 28-37 with a 4.42 ERA and 553 strikeouts in 532 innings of work.
In his one season with the Hooks in 2008, a season fraught with blister and shoulder issues, Norris was 3-8 with a 4.05 ERA in 19 starts. So staying healthy is a key for Norris, especially with the additional responsibilities that have been foisted upon his plate.
"When I got to the big leagues, like I said, we were such an old team and I was just trying to fit in and be a part of the group and learn from the older players," Norris said. "Now I've definitely been pushed into that role to be one of the older guys.
"Hopefully from what they had taught me and what I learned from them I can carry over to these young guys and kind of take that veteran leadership a little bit more, and it's something I look forward to," Norris said. "But by the same token, actions speak louder than words and you need to get your work done and take care of that."
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WeatherBug for Samsung Bada
Pillsbury, ND 58065
WeatherBug Mobile
Get local current conditions, extended forecast, radar and satellite images, severe weather alerts and more on your Samsung smartphone with WeatherBug. To download, simply access the Samsung Apps store via the icon on your Bada OS device and search using the keyword "WeatherBug" or by visiting the "News" category.
Standard Features:
- Access to the WeatherBug Network — the source for truly live, local weather with the largest network of professional weather stations in the U.S. and thousands of locations around the world.
- Extended 7-day and detailed forecasts
- Radar animation*
- Severe weather alerts (NWS alerts)*
- Three (3) enhanced contour map overlays: Temperature, Radar* and Satellite
- Weather camera images and time-lapse animation*
- Unlimited saved locations
- View weather in Metric or English Imperial (U.S.) units
* U.S. locations only
Press Releases
WeatherBug Featured Products
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California Crews Hold Ground Against Blaze That Jumped Line
California fire officials say a wildfire burning east of Lower Lake has grown to more than 4 and half square miles in a few…More >
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Friday's full moon will be the second full moon of July, a fairly unusual phenomenon that has come to be known as 'a blue…More >
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You too can help save our planet and put money back in your wallet. Learn how you can take the first steps to reduce your environmental impact, including driving green, easy ways you can conserve water, and energy saving tips. To learn more and discover the benefits of going green, visit WeatherBug's green living section. More >
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Get faster alerts and better forecasts from the exclusive neighborhood-level WeatherBug network.Learn More


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Although the National Football Museum's move to Manchester (from Preston) has not been without controversy, it seems fitting that Manchester, a city known the world over for its footballing prowess, will soon be its home.
Due to open in late 2011, the museum holds both the FIFA and FA collections, including the hallowed ball used in that World Cup final (1966, if you must ask), while changing exhibitions will add interest to permanent displays. The museum itself is housed inside Urbis, once Manchester's ill-fated 'museum of the city', a striking glass building that is being given an £8 million overhaul in readiness for its latest incarnation as the home of British football.
Area City Centre
Transport Metrolink Victoria, or City Centre buses, or Victoria rail
Open see website for details
Admission see website for details


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MoSync apps run on Symbian^3Published by Abi Waqas on November 04, 2010
We, at MoSync, have taken the leverage to run MoSync apps on the newly launched Symbian 3 device : Nokia N8-00 and guess, what the result would be?
MoSync in South KoreaPublished by Alex Jonsson on October 13, 2010
MoSyncers Patrick Broman and Alex Jonsson are on a developer road trip to South Korea, on Tuesday holding a one-day MoSync seminar at KOVA in Guro-gu, Seoul, followed by meetings and discussion with Korean companies on mobile application development and the local market situation in the region.
MoSync C++ seamless native development for Android phonesPublished by Antony Hartley on October 06, 2010
The latest version of MoSync (that's 2.4) contains support for building native C++ applications on Android.A few people have asked me, whats the point, why not use the Android NDK to write C++ ? I'll omit of the obvious benefits of MoSync, such as the truly cross-platform-ness, the same code working on lots of different devices, screen size and OS's, etc.
MoSync Smartphone Device CoveragePublished by Antony Hartley on October 06, 2010
We've been working hard at MoSync, so now I'm able to publish some stats about our device coverage IPhone and IPad - MoSync support all of these, so thats another 4-5 devices, but with a reasonable market share. Android Devices - Their are Approx 100 Different Android devices 90% of them are v1.6 and above, these devices are suitable for MoSync.
MoSync project TeDuCo wins first prize in Cut The Wire awardsPublished by Alex Jonsson on September 24, 2010
BTH Innovation hands out BTH Innovation Prize at this year's Cut The Wire Award ceremony. The winner is: MoSync and Astando with Teduco Jury motivation: By combining new technology with old, by creating new and well adapted data and by focusing on a very distinct end-user
Location Starter App -- video and source codePublished by Alex Jonsson on September 10, 2010
Location. Location. Location!These three words managed to define the real estate boom of our age. But now the same three words can be used to define the 'next' big wave of personalized services for smartphone users, services that revolve around not only what you want and like, but also where you are.The nearest restaurant, the bus stations, must visit places for tourists...GPS (Global Positioning Systems...but you knew that right?) has now changed the way we interact with the world.
StringBuilder for MoSyncPublished by Sam Pickard on September 05, 2010
In C++, strings have traditionally been single byte char arrays. Contiguous memory is allocated for the length of the string you want. You can then pass a char* to the first character to pass a string around. The length of the string is then calculated by counting the characters until you get to a null character (0x000000). This is very handy in the C++ way of low level memory access but has two inherent flaws. Firstly, it doesn't really support multi-byte character sets, and secondly, its easy to write more data into the string that you've allocated.
Porting Lua, TinyJS and Webkit - An Interview with Mikael KindborgPublished by Chris Hughes on August 17, 2010
Mikael Kindborg is well known in the dynamic languages and Smalltalk communities. He's a former lecturer in Computer Science at Linköping University, and he's been working recently with porting Lua, TinyJS and WebKit to MoSync. I took the opportunity of a break in his busy schedule to ask him about this work.
Question: Do intermediate layers always produce substandard applications? Answer: NoPublished by Henrik von Schoultz on June 30, 2010
This blog post was originally written by Patrick Broman and explains how powerful and important a cross platform framework is when you produce Mobile applications. The Gartner group rates cross platform development tools very important to reach out and maintain Mobile Applications.
Serial Data AccessPublished by Sam Pickard on May 27, 2010
I've recently been writing a tutorial on storing and reading data on the device in MoSync. For part of the tutorial, I wanted to use some classes which I'd already written which allow you serial access to your data, and are an extension to the included DataHandler class.


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We welcome parents and students on August 25, 2014
We wear navy blue bottoms and white tops daily at CCB!
It is with great pleasure that our teachers and staff welcome you to the Charles Carroll Barrister Elementary School Website. We are excited about the start of the new school year and the opportunity to work with our children. As the school year begins, the staff is committed to our school's vision to empower students with knowledge, values and high expectations for a successful college and career-ready future. Our goal is to create self-sufficient/independent citizens with an enthusiasm for learning and respect for self and others. Together we will strive for excellence for all our students.


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Most popular at the top
Firebrand's LadyPocket Books 2010; US$ 4.99
A Simon & Schuster eBook. Simon & Schuster has a great book for every reader. more...
Child-Focused PracticeKarnac Books 1998; US$ 27.95
The author, with over twenty years of experience of working with children, writes refreshingly about the practical aspects of his work. He takes traditional and contemporary theories and explains them in the context of how he works with children. more...
The Performance of PracticeKarnac Books 2007; US$ 27.95
'This book is about how to maintain an aliveness to the possibilities in therapy and practice and how to challenge ideas of orthodoxy in theory and methodologies that can become stale or followed like religions. The central metaphor is the performance of practice emphasized in the spoken word and expressed in all its non-verbal complexity. How we,... more...
Nazi PrincessThe History Press 2011; US$ 26.24
Born to a middle-class Viennese family and of partly Jewish descent, after marriage to (and divorce from) a German prince, Stephanie von Hohenlohe became a close confidante of Hitler, Goring, Himmler (who declared her an 'honorary Aryan') and von Ribbentrop. After arriving in London in 1932, she moved in the most exclusive circles, arranging the visits... more...
Firebrand's LadyPocket Books 2010; Not Available
A Simon & Schuster eBook. Simon & Schuster has a great book for every reader. more...
Firebrand's LadyPocket Books 2010; Not Available
A Simon & Schuster eBook. Simon & Schuster has a great book for every reader. more...
Britain on the BrinkPen and Sword 2012; US$ 8.99
At the time of the Cuban Missile Crisis, Britain was Americas first-line defense, a vulnerable, but unsinkable aircraft carrier on which the United States based the Strategic Air Commands first-strike elements of their Americas nuclear deterrent. The Strategic Air Commands UK bases and the RAFs V-Force were... more...
Creating Dynamic UI with Android FragmentsPackt Publishing 2013; US$ 23.99
A fast-paced tutorial that guides you through everything you need to know about dynamic UI design for Android devices.This book is for developers with a basic understanding of Android programming who would like to improve the appearance and usability of their applications. Whether you?re looking to create a more interactive user experience, create... more...
Bulletproof Flowers for the SouthTaylor Trade Publishing 1999; US$ 31.99
The former co-host of the PBS show The Victory Garden illustrates how to plant, grow, and care for a variety of heat-resistant Southern flowers. more...
Launch Pad UKPen and Sword 2009; US$ 10.99
For most British people the weekend of 27/28 October 1962 could so very easily have been their last weekend on earth, yet astonishingly the fact that Britain's nuclear deterrent forces went to an unprecedented level of readiness was kept secret from the public. Thor nuclear-tipped ballistic missiles stood on a round-the-clock wartime state of alert... more...


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Lingerie and football, together at last! The Legends Football League is home to the best female athletes in the sexiest outfits, and we're here to give you a rundown of some of the best the league has to offer in both departments.
- VH1 Announces Premiere Date For The Game's Dating Show "She's Got Game"501 Views7 Comments
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- Fetty Wap The First Male Rapper To Have Two Top 10 Singles Since Lil Wayne17,937 Views115 Comments
- Damian Lillard Releases New Song Using Classic Jadakiss Beat1,468 Views0 Comments
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BT and GENBAND upgrade BT's iComms UC service
BT and GENBAND jointly announced the latest upgrades to BT's iComms Unified Communication service, which provides businesses with a variety of carrier-hosted IP communications solutions aim at enhancing how people do business.
Powered by GENBAND's carrier-hosted offering, the new upgrade allows BT to provide Centrex IP and SIP services to customers. The new upgrades to the service include advanced voice and multimedia collaboration services such as unified communications, real-time presence and customized Web portal capabilities. Customers can now launch a voice conference with a personal conference bridge and run web meetings on their personal web collaboration tool while sharing their desktop.
For more:
- read the release
Related news:
GENBAND announces roadmap and GENiUS platform
Genband announces post-Nortel CVAS merger moves
BT's Onevoice gets Ribbit up and running


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Brace yourself, because for this Twistys LIVE, we've got the one and only Capri Cavanni going hardcore for all of her loyal Twistys fans! Watch her tease, pose, and strip down to nothing before taking Xander Corvus's big dick deep in her perfect pink pussy!
Come lie by the pool with our hottest teen starlets Abigail Mac, Ryan Ryans, and Samantha Rone, as they put on another steamy live show for you! Watch as these horny babes answered fan requests, showed off their gorgeous, tanned bodies, and broke out the strap-on to satisfy their desire for naughty lesbian sex in the shallow water.
Brace yourself for some oily action as the busty and beautiful Nicole Aniston welcomes her sexy friends, Twistys Treats Dillion Harper and Madison Ivy, to celebrate being named Twistys Treat of the Year for 2014. Watch as the three lovely ladies rub down each and every inch of each other's gorgeous bodies LIVE! There's vibrators, strap-ons, and lots and lots of pretty pink pussy as three of the sexiest Twistys Treats ever cum hard all for your viewing pleasure!
coming soon
It's two busty babes in sexy stockings as Twisty's Treat of the Year Mia Malkova has a masturbation competition with Treat of the Month Nicole Aniston. Watch these two lovely ladies see who can cum the most in this wet and wild edition of Twisty's LIVE!


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Below is the poem entitled "Look Me In The Eyes, Tell Me How Many I Killed" which was written by poet
Ortiz. Please feel free to comment on this poem. However, please remember, PoetrySoup is a place of encouragement and growth.
Read Poems by
Derek Ortiz
Come on, shut up already, you stand clear to confusion, not of my business, i multiply
myself to vanquish, you pray to god for power and love? I pray to god for my life to be
brought to live, don't tell me you said slave, I don't believe your kind of lie, so clear
now that you taken all from us, myself and my only self, blood stood tall to breath lies,
i can still sense you here but i can't see you, i smell your perfume, but i can't see you,
like a ghost you came in im sure of, you lied to us all, took our loved one's, left us in
ruins and walk away like a victor! I refuse! I will stand strong too, this is my fight, i
don't need a man to help me when im the only men who lost it all, partner in crime we all
been fighting trought, your not gonna tell me another lie, nevertheless i will stand to my
ground and won't let go of their rotten corpses, they could be dead now, but i will keep
'em there for you to see what you did, her face is rot, smells like the hell once brought
upon your soul and feel's like no more soldier will stand to die here, no sense fall, no
sense fail, i can still feel you here but i can't see you, so i must be dead to see? Then
with this bullet i will garanty my eye's one last fall upon your dirty soul, massacre, i
will to your past, im not the toy you were told to play with, im the man who will disobey
god to destroy who you are!


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Tools for Developing Knowledge
Peer Group Program
Peer Advisor Program
Student-Run Toolbox Committees
My Props INCENTIVE Programs
LINKS Mentoring Program
Student Leadership Institute
University Leadership Conference
Chicano Resource Center
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1/4" to 1/4". 14 AWG.
Live Wire cables have the strength and durability to withstand more than everyday use.
Superior wire
Your cable is your sound. Superior wiring allows your signal to be quiet, eliminating frustrating hums, buzzing, and crackles.
Sturdy construction
Sturdy construction enhances signal and performance. Flexible design eliminates kinks and twists for a tangle-free performance whether you're in the studio or on the stage.
Livewire Advantage 14GA Speaker Cable 25 ft.
Item # 110002847 | Condition: Info | Customer Ratings: ( 5 Based on 4 reviews)
- Store
- Availability
Usually arrives in 3-5 Days
3 on hand
Protect Your Gear
Accidental damage, normalNormal wear and power surges are no longer an issue with these comprehensive and affordable plans. We even pay to ship your gear for repairs. More details.
Covers much more than the original manufacturer's warranty:
Limit of Liability: The least of the cost of (1) authorized repairs, (2) replacement with a product of similar features, (3) reimbursement for authorized repairs or replacement or (4) the price that you paid for the product.
*Limitations and exclusions apply. See terms and conditions for program details
1/4" to 1/4". 14 AWG.
As reviewed by on
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Sweet CableAs reviewed by Murderface on 10/1/2008
Dude, this cable is probably one of the best cables I have. I use mine all the time. Its the best cable I have.15 of 26 people found this review helpful.Was this review helpful?Thank You for rating this review!
Good bang for your buckAs reviewed by Richard on 7/30/2008
Works well for my application. It's not coaxial (shielded) wire, its just two wires incased in a round sleeve so it's easy to cut and solder if you want to shorten it up or change ends.22 of 23 people found this review helpful.Was this review helpful?Thank You for rating this review!
Sound is in Your CablesAs reviewed by Brandy on 4/27/2010
The saying that your sound is in your cables is true. These were used to replace old cables, and they were the only thing we replaced. We thought our speakers were blown, but decided to start replacing the cheapest things first to fix the issue before just buying new speakers. Our speakers went from sounding blown to being crisp and clear.9 of 11 people found this review helpful.Was this review helpful?Thank You for rating this review!
- Product DetailsCustomer reviewsPrice
For your convenience, our online inventory has been expanded to include items in our retail locations. Some items may be fulfilled from Guitar Center retail stores*. Items will be inspected to ensure the store has the product you want, in the condition you expect. If not, a store manager will contact you by phone or email within one hour.
*Select items are available for in store pick-up only