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Open practice run-throughs are open ONLY TO HANDLERS AND DOGS WITH BARN HUNT EXPERIENCE. Experience includes attendance in one of my classes, attendance in classes taught elsewhere, or BHA trial experience. Open practices do not include rat familiarization or discussions and are therefore not intended to serve as a Barn Hunt Introduction. Run-throughs are structured like a trial with a judge inside the ring. Training in the ring is allowed, but training advice by the judge will be limited. All handlers/dogs will be expected to exit the ring when their time is called, and dogs that eliminate in the ring will be immediately excused from that run.
Each run-through is $5.
No sign up or preregistration available; run-throughs are first come, first served.
Upcoming Practice Days
Handlers are responsible for the action of their dogs at all times. In the event a dog (or handler) harms a rat in any way, the handler will pay $30 to replace the rat. If the action is deemed blatant, the team will be asked to leave immediately.
Furthermore, in an effort to protect our equipment (straw bales) during open practice and all advanced classes, handlers will be financially responsible, in addition to be excused from the ring, in the event their dog eliminates (defined as urinates, defecates, or vomits) anywhere in the ring. Handlers will pay $8, due immediately, for each elimination incident.


text:
BBC Sport - England v West Indies: Stuart Broad praises Ashley Giles
England have gone back to basics - Broad
- From the section Cricket
England's captain Stuart Broad praises the "relaxed" approach of new coach Ashley Giles ahead of England's one-day international series against West Indies.
The Nottinghamshire all-rounder says the new coach has "gone back to basics" as England look to rebuild following a disastrous Ashes series that culminated in the exit of coach Andy Flower and batsmen Kevin Pietersen.
Broad says it is "great" that Jonathan Trott is due to return to the game, as England play three ODIs and three Twenty20 in preparation for the ICC Twenty20 World Cup in Bangladesh in March.
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|Publication number
|Publication type
|Application number
|Publication date
|Oct 5, 2010
|Filing date
|May 8, 2007
|Priority date
|Sep 25, 2003
|Fee status
|Also published as
|Publication number
|Stanislaw Flasinski
|Original Assignee
|Monsanto Technology Llc
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (15), Non-Patent Citations (25), Classifications (17), Legal Events (1)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a Divisional of U.S. patent application Ser. No. 10/950,233, filed 24 Sep. 2004 now U.S. Pat. No. 7,408,054 and published as U.S. Patent Application Publication US20060162010A1, which claims priority to U.S. Provisional Patent Application No. 60/505,949, filed 25 Sep. 2003, both of which are incorporated by reference in their entirety herein.
Two copies of the sequence listing (Seq. Listing Copy 1 and Seq. Listing Copy 2) and a computer-readable form of the sequence listing, all on CD-ROMs, each containing the file named pa—01298.rpt, which is 16,783 bytes (measured in MS Windows�) and was created on May 5, 2007, are hereby incorporated by reference.
The invention relates to the field of plant molecular biology and plant genetic engineering and polynucleotide molecules useful for the expression of transgenes in plants.
One of the goals of plant genetic engineering is to produce plants with agronomically desirable characteristics or traits. The proper expression of a desirable transgene in a transgenic plant is one way to achieve this goal. Regulatory elements such as promoters, leaders, and introns are non-coding polynucleotide molecules which play an integral part in the overall expression of genes in living cells. Isolated regulatory elements that function in plants are therefore useful for modifying plant phenotypes through the methods of genetic engineering.
Many regulatory elements are available and are useful for providing good overall expression of a transgene. For example, constitutive promoters such as P-FMV, the promoter from the 35S transcript of the Figwort mosaic virus (U.S. Pat. No. 6,051,753); P-CaMV 35S, the promoter from the 35S RNA transcript of the Cauliflower mosaic virus (U.S. Pat. No. 5,530,196); P-Rice Actin 1, the promoter from the actin 1 gene of Oryza sativa (U.S. Pat. No. 5,641,876); and P-NOS, the promoter from the nopaline synthase gene of Agrobacterium tumefaciens are known to provide some level of gene expression in most or all of the tissues of a plant during most or all of the plant's lifespan. While previous work has provided a number of regulatory elements useful to affect gene expression in transgenic plants, there is still a great need for novel regulatory elements with beneficial expression characteristics. Many previously identified regulatory elements fail to provide the patterns or levels of expression required to fully realize the benefits of expression of selected genes in transgenic crop plants.
Spatial organization within the eukaryotic cell and directed movements of the cell contents are mediated by the cytoskeleton, a network of filamentous protein polymers that permeates the cytosol. Actin is one of the three major families of proteins making up the cytoskeleton. Members of this multi-gene family have been reported in almost all eukaryotic species including yeast, humans, mouse, Drosophila, tobacco, maize, rice, soybean, potato and Arabidopsis. Plant actins are encoded by a multi-gene family, constituted by a number of different isotypes.
We hypothesized that the regulatory elements from an actin gene might have a constitutive expression pattern and that the regulatory elements could be useful to direct expression of a transgene such as a glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene to produce a glyphosate tolerant plant. The efficient production of glyphosate tolerant plants requires the use of regulatory elements capable of directing transgene expression in all tissues including the most sensitive reproductive organs such as anthers and meristem tissues. The present invention thus provides such regulatory elements isolated from actin genes of Oryza sativa and Zea mays.
In one embodiment the invention provides polynucleotide molecules isolated from Oryza sativa and Zea mays useful for modulating transgene expression in plants. In another embodiment the invention provides expression constructs containing the polynucleotide molecules useful for modulating transgene expression in plants. In another embodiment the invention provides transgenic plants and seeds containing the polynucleotide molecules useful for modulating transgene expression in plants.
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
The invention disclosed herein provides polynucleotide molecules having gene regulatory activity from Oryza sativa and Zea mays. The design, construction, and use of these polynucleotide molecules are one object of this invention. The polynucleotide sequences of these polynucleotide molecules are provided as SEQ ID NO: 1-7. These polynucleotide molecules are capable of affecting the transcription of operably linked transcribable polynucleotide molecules in both vegetative and reproductive tissues of plants and therefore can selectively regulate expression of transgenes in these tissues.
As used herein, the term "polynucleotide molecule" refers to the single- or double-stranded DNA or RNA of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide bases, respectively, read from the 5′ (upstream) end to the 3′ (downstream) end.
As used herein, the term "polynucleotide sequence" refers to the sequence of a polynucleotide molecule. The nomenclature for DNA bases as set forth at 37 CFR �1.822 is used.
As used herein, the term "gene regulatory activity" refers to a polynucleotide molecule capable of affecting transcription or translation of an operably linked transcribable polynucleotide molecule. An isolated polynucleotide molecule having gene regulatory activity may provide temporal or spatial expression or modulate levels and rates of expression of the operably linked transcribable polynucleotide molecule. An isolated polynucleotide molecule having gene regulatory activity may comprise a promoter, intron, leader, or 3′ transcriptional termination region.
As used herein, the term "regulatory element" refers to an isolated polynucleotide molecule capable of having gene regulatory activity. A regulatory element may comprise a promoter, intron, leader, or 3′ transcriptional termination region.
As used herein, the term "promoter" refers to a polynucleotide molecule that is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A "plant promoter" is a native or non-native promoter that is functional in plant cells. A plant promoter can be used as a 5′ regulatory element for modulating expression of an operably linked gene or genes. Plant promoters may be defined by their temporal, spatial, or developmental expression pattern.
A promoter comprises subfragments that have promoter activity. Subfragments may comprise enhancer domains and may be useful for constructing chimeric promoters. Subfragments of SEQ ID NO: 1 comprise at least about 75, 85, 90, 95, 110, 125, 250, 400, 750, 1000, 1300, 1500, and 1700 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 1-7. Subfragments of SEQ ID NO: 2 comprise at least about 95, 110, 125, 250, 400, 750, 1000, and 1300 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 2. Subfragments of SEQ ID NO: 3 comprise at least about 95, 110, 125, 250, 400, 750, 1000, 1300, 1500, and 1800 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 3. Subfragments of SEQ ID NO: 4 comprise at least about 95, 110, 125, 250, 400, 750, and 1000 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 4. Subfragments of SEQ ID NO: 5 comprise at least about 95, 110, 125, 250, 400, 750, 1000, 1300, 1500, 1800, and 2500 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 5. Subfragments of SEQ ID NO: 6 comprise at least about 95, 110, 125, 250, 400, 750, 1000, 1300, and 1500 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 6. Subfragments of SEQ ID NO: 7 comprise at least about 95, 110, 125, 250, 400, 750, 1000, and 1300 contiguous nucleotides of the polynucleotide sequence of SEQ ID NO: 7.
As used herein, the term "enhancer domain" refers to a cis-acting transcriptional regulatory element, a.k.a. cis-element, which confers an aspect of the overall control of gene expression. An enhancer domain may function to bind transcription factors, trans-acting protein factors that regulate transcription. Some enhancer domains bind more than one transcription factor, and transcription factors may interact with different affinities with more than one enhancer domain. Enhancer domains can be identified by a number of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5′ end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional DNA sequence comparison methods. The fine structure of an enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods. Enhancer domains can be obtained by chemical synthesis or by isolation from promoters that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Thus, the design, construction, and use of enhancer domains according to the methods disclosed herein for modulating the expression of operably linked polynucleotide molecules are encompassed by the present invention.
As used herein, the term "chimeric" refers to the product of the fusion of portions of two or more different polynucleotide molecules. As used herein, the term "chimeric promoter" refers to a promoter produced through the manipulation of known promoters or other polynucleotide molecules. Such chimeric promoters may combine enhancer domains that can confer or modulate gene expression from one or more promoters, for example, by fusing a heterologous enhancer domain from a first promoter to a second promoter with its own partial or complete regulatory elements. Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein for modulating the expression of operably linked polynucleotide molecules are encompassed by the present invention.
As used herein, the term "percent sequence identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference polynucleotide molecule (or its complementary strand) as compared to a test polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG� Wisconsin Package� (Accelrys Inc., Burlington, Mass.). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence.
As used herein, the term "substantial percent sequence identity" refers to a percent sequence identity of at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity. Thus, one embodiment of the invention is a polynucleotide molecule that has at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 98% or about 99% sequence identity with a polynucleotide sequence described herein. Polynucleotide molecules that are capable of regulating transcription of operably linked transcribable polynucleotide molecules and have a substantial percent sequence identity to the polynucleotide sequences of the polynucleotide molecules provided herein are encompassed within the scope of this invention.
Promoter Isolation and Modification Methods
Any number of methods well known to those skilled in the art can be used to isolate fragments of a promoter disclosed herein. For example, PCR (polymerase chain reaction) technology can be used to amplify flanking regions from a genomic library of a plant using publicly available sequence information. A number of methods are known to those of skill in the art to amplify unknown polynucleotide molecules adjacent to a core region of known polynucleotide sequence. Methods include but are not limited to inverse PCR (IPCR), vectorette PCR, Y-shaped PCR, and genome walking approaches. Polynucleotide fragments can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer. For the present invention, the polynucleotide molecules were isolated from genomic DNA by designing PCR primers based on available sequence information.
Novel chimeric promoters can be designed or engineered by a number of methods. For example, a chimeric promoter may be produced by fusing an enhancer domain from a first promoter to a second promoter. The resultant chimeric promoter may have novel expression properties relative to the first or second promoters. Novel chimeric promoters can be constructed such that the enhancer domain from a first promoter is fused at the 5′ end, at the 3′ end, or at any position internal to the second promoter. The location of the enhancer domain fusion relative to the second promoter may cause the resultant chimeric promoter to have novel expression properties relative to a fusion made at a different location.
Those of skill in the art are familiar with the standard resource materials that describe specific conditions and procedures for the construction, manipulation, and isolation of macromolecules (e.g., polynucleotide molecules, plasmids, etc.), as well as the generation of recombinant organisms and the screening and isolation of polynucleotide molecules.
As used herein, the term "construct" refers to any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a polynucleotide molecule where one or more polynucleotide molecule has been operably linked.
As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a gene of interest, where the polynucleotide molecules are so arranged that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may be part of a single contiguous polynucleotide molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
As used herein, the term "transcribable polynucleotide molecule" refers to any polynucleotide molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable polynucleotide molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3 (2000) J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press.
Constructs of the present invention would typically contain a promoter operably linked to a transcribable polynucleotide molecule operably linked to a 3′ transcription termination polynucleotide molecule. In addition, constructs may include but are not limited to additional regulatory polynucleotide molecules from the 3′-untranslated region (3′ UTR) of plant genes (e.g., a 3′ UTR to increase mRNA stability of the mRNA, such as the PI-II termination region of potato or the octopine or nopaline synthase 3′ termination regions). Constructs may include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA polynucleotide molecule which can play an important role in translation initiation and can also be a genetic component in a plant expression construct. For example, non-translated 5′ leader polynucleotide molecules derived from heat shock protein genes have been demonstrated to enhance gene expression in plants (see for example, U.S. Pat. Nos. 5,659,122 and 5,362,865, all of which are hereby incorporated by reference). These additional upstream and downstream regulatory polynucleotide molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
Thus, one embodiment of the invention is a promoter such as provided in SEQ ID NO: 1-7, operably linked to a transcribable polynucleotide molecule so as to direct transcription of said transcribable polynucleotide molecule at a desired level or in a desired tissue or developmental pattern upon introduction of said construct into a plant cell. In some cases, the transcribable polynucleotide molecule comprises a protein-coding region of a gene, and the promoter provides for transcription of a functional mRNA molecule that is translated and expressed as a protein product. Constructs may also be constructed for transcription of antisense RNA molecules or other similar inhibitory RNA in order to inhibit expression of a specific RNA molecule of interest in a target host cell.
Exemplary transcribable polynucleotide molecules for incorporation into constructs of the present invention include, for example, polynucleotide molecules or genes from a species other than the target gene species, or even genes that originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. Exogenous gene or genetic element is intended to refer to any gene or polynucleotide molecule that is introduced into a recipient cell. The type of polynucleotide molecule included in the exogenous polynucleotide molecule can include a polynucleotide molecule that is already present in the plant cell, a polynucleotide molecule from another plant, a polynucleotide molecule from a different organism, or a polynucleotide molecule generated externally, such as a polynucleotide molecule containing an antisense message of a gene, or a polynucleotide molecule encoding an artificial or modified version of a gene.
The promoters of the present invention can be incorporated into a construct using marker genes as described and tested in transient analyses that provide an indication of gene expression in stable plant systems. As used herein the term "marker gene" refers to any transcribable polynucleotide molecule whose expression can be screened for or scored in some way. Methods of testing for marker gene expression in transient assays are known to those of skill in the art. Transient expression of marker genes has been reported using a variety of plants, tissues, and DNA delivery systems. For example, types of transient analyses can include but are not limited to direct gene delivery via electroporation or particle bombardment of tissues in any transient plant assay using any plant species of interest. Such transient systems would include but are not limited to electroporation of protoplasts from a variety of tissue sources or particle bombardment of specific tissues of interest. The present invention encompasses the use of any transient expression system to evaluate promoters or promoter fragments operably linked to any transcribable polynucleotide molecules, including but not limited to selected reporter genes, marker genes, or genes of agronomic interest. Examples of plant tissues envisioned to test in transients via an appropriate delivery system would include but are not limited to leaf base tissues, callus, cotyledons, roots, endosperm, embryos, floral tissue, pollen, and epidermal tissue.
Any scorable or screenable marker gene can be used in a transient assay. Exemplary marker genes for transient analyses of the promoters or promoter fragments of the present invention include a GUS gene (U.S. Pat. No. 5,599,670, hereby incorporated by reference) or a GFP gene (U.S. Pat. Nos. 5,491,084 and 6,146,826, both of which are hereby incorporated by reference). The constructs containing the promoters or promoter fragments operably linked to a marker gene are delivered to the tissues and the tissues are analyzed by the appropriate mechanism, depending on the marker. The quantitative or qualitative analyses are used as a tool to evaluate the potential expression profile of the promoters or promoter fragments when operatively linked to genes of agronomic interest in stable plants.
Thus, in one preferred embodiment, a polynucleotide molecule of the present invention as shown in SEQ ID NO: 1-7 is incorporated into a DNA construct such that a polynucleotide molecule of the present invention is operably linked to a transcribable polynucleotide molecule that provides for a selectable, screenable, or scorable marker. Markers for use in the practice of the present invention include, but are not limited to transcribable polynucleotide molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), proteins that confer antibiotic resistance, or proteins that confer herbicide tolerance. Useful antibiotic resistance markers, including those encoding proteins conferring resistance to kanamycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec/strep) and gentamycin (aac3 and aacC4) are known in the art. Herbicides for which transgenic plant tolerance has been demonstrated and the method of the present invention can be applied, include but are not limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase inhibitors, and isoxasflutole herbicides. Polynucleotide molecules encoding proteins involved in herbicide tolerance are known in the art, and include, but are not limited to a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) described in U.S. Pat. Nos. 5,627,061, 5,633,435, 6,040,497 and in U.S. Pat. No. 5,094,945 for glyphosate tolerance, all of which are hereby incorporated by reference; polynucleotides encoding a glyphosate oxidoreductase and a glyphosate-N-acetyl transferase (GOX, U.S. Pat. No. 5,463,175 and GAT, U.S. Patent publication 20030083480, herein incorporated by reference); a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) described in U.S. Pat. No. 4,810,648 for Bromoxynil tolerance, which is hereby incorporated by reference; a polynucleotide molecule encoding phytoene desaturase (crtI) described in Misawa et al, (1993) Plant J. 4:833-840 and Misawa et al, (1994) Plant J. 6:481-489 for norflurazon tolerance; a polynucleotide molecule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res. 18:2188-2193 for tolerance to sulfonylurea herbicides; and the bar gene described in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for glufosinate and bialaphos tolerance; resistant hydroxyphenyl pyruvate dehydrogenase (HPPD, U.S. Pat. No. 6,768,044). The promoter of the present invention can express genes that encode for phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hydroxyphenyl pyruvate dehydrogenase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase, glyphosate oxidoreductase and glyphosate-N-acetyl transferase.
Where plastid targeting is necessary, for example, the EPSPS enzyme functions in a plant chloroplast, therefore, DNA molecules encoding a chloroplast transit peptide (CTP) are engineered into a DNA molecule encoding an EPSPS protein to encode a fusion protein of the CTP to the N terminus of an EPSPS creating a chimeric molecule. A chimeric polynucleic acid coding sequence is comprised of two or more open reading frames joined in-frame that encode a chimeric protein, for example, a chloroplast transit peptide and an EPSPS enzyme. A chimeric gene refers to the multiple genetic elements derived from heterologous sources operably linked to comprise a gene. In the present invention the DNA construct expresses a chimeric CTP-EPSPS protein that directs the glyphosate resistant EPSPS protein into the plant chloroplast. In a native plant EPSPS gene, chloroplast transit peptide regions are contained in the native coding sequence (for example, CTP2, Klee et al., Mol. Gen. Genet. 210:47-442, 1987). The CTP is cleaved from the EPSPS enzyme at the chloroplast membrane to create a "mature EPSPS or EPSPS enzyme" that refers to the polypeptide sequence of the processed protein product remaining after the chloroplast transit peptide has been removed. The production of glyphosate tolerant plants by expression of a fusion protein comprising an amino-terminal CTP with a glyphosate resistant EPSPS enzyme is well known by those skilled in the art, (U.S. Pat. Nos. 5,627,061, 5,633,435, 5,312,910, EP 0218571, EP 189707, EP 508909, and EP 924299). Those skilled in the art will recognize that various chimeric constructs can be made that utilize the functionality of a particular CTP to import glyphosate resistant EPSPS enzymes into the plant cell chloroplast.
In one embodiment of the invention, a polynucleotide molecule as shown in SEQ ID NO: 1-7 is incorporated into a construct such that a polynucleotide molecule of the present invention is operably linked to a transcribable polynucleotide molecule that is a gene of agronomic interest. As used herein, the term "gene of agronomic interest" refers to a transcribable polynucleotide molecule that includes but is not limited to a gene that provides a desirable characteristic associated with plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. The expression of a gene of agronomic interest is desirable in order to confer an agronomically important trait. A gene of agronomic interest that provides a beneficial agronomic trait to crop plants may be, for example, including, but not limited to genetic elements comprising herbicide resistance (U.S. Pat. Nos. 5,633,435 and 5,463,175), increased yield (U.S. Pat. No. 5,716,837), insect control (U.S. Pat. Nos. 6,063,597; 6,063,756; 6,093,695; 5,942,664; and 6,110,464), fungal disease resistance (U.S. Pat. Nos. 5,516,671; 5,773,696; 6,121,436; 6,316,407, and 6,506,962), virus resistance (U.S. Pat. Nos. 5,304,730 and 6,013,864), nematode resistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S. Pat. No. 5,516,671), starch production (U.S. Pat. Nos. 5,750,876 and 6,476,295), modified oils production (U.S. Pat. No. 6,444,876), high oil production (U.S. Pat. Nos. 5,608,149 and 6,476,295), modified fatty acid content (U.S. Pat. No. 6,537,750), high protein production (U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition (U.S. Pat. Nos. 5,985,605 and 6,171,640), biopolymers (U.S. Pat. No. 5,958,745 and U.S. Patent Publication No. US20030028917), environmental stress resistance (U.S. Pat. No. 6,072,103), pharmaceutical peptides (U.S. Pat. No. 6,080,560), improved processing traits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzyme production (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), and biofuel production (U.S. Pat. No. 5,998,700). The genetic elements, methods, and transgenes described in the patents listed above are hereby incorporated by reference.
Alternatively, a transcribable polynucleotide molecule can effect the above mentioned phenotypes by encoding a RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense, inhibitory RNA (RNAi), or cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product. Thus, any polynucleotide molecule that encodes a protein or mRNA that expresses a phenotype or morphology change of interest may be useful for the practice of the present invention.
The constructs of the present invention are generally double Ti plasmid border DNA constructs that have the right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmid isolated from Agrobacterium tumefaciens comprising a T-DNA, that along with transfer molecules provided by the Agrobacterium cells, permits the integration of the T-DNA into the genome of a plant cell. The constructs also contain the plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, or LBA4404, however, other strains known to those skilled in the art of plant transformation can function in the present invention.
Transformed Plants and Plant Cells
As used herein, the term "transformed" refers to a cell, tissue, organ, or organism into which has been introduced a foreign polynucleotide molecule, such as a construct. The introduced polynucleotide molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced polynucleotide molecule is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a foreign polynucleotide molecule. A plant transformation construct containing a promoter of the present invention may be introduced into plants by any plant transformation method. Methods and materials for transforming plants by introducing a plant expression construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated methods including electroporation as illustrated in U.S. Pat. No. 5,384,253; microprojectile bombardment as illustrated in U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865; Agrobacterium-mediated transformation as illustrated in U.S. Pat. Nos. 5,824,877; 5,591,616; 5,981,840; and 6,384,301; and protoplast transformation as illustrated in U.S. Pat. No. 5,508,184, all of which are hereby incorporated by reference.
Methods for specifically transforming dicots are well known to those skilled in the art. Transformation and plant regeneration using these methods have been described for a number of crops including, but not limited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut (Arachis hypogaea), and members of the genus Brassica.
Methods for transforming monocots are well known to those skilled in the art. Transformation and plant regeneration using these methods have been described for a number of crops including, but not limited to, barley (Hordeum vulgarae); maize (Zea mays); oats (Avena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticum aestivum), and alfalfa (Medicago sativa). It is apparent to those of skill in the art that a number of transformation methodologies can be used and modified for production of stable transgenic plants from any number of target crops of interest.
The transformed plants are analyzed for the presence of the genes of interest and the expression level and/or profile conferred by the promoters of the present invention. Those of skill in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays.
The seeds of this invention can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plant lines comprising the construct of this invention and expressing a gene of agronomic interest.
The present invention also provides for parts of the plants of the present invention. Plant parts, without limitation, include seed, endosperm, ovule and pollen. In a particularly preferred embodiment of the present invention, the plant part is a seed.
Still yet another aspect of the invention is a method of inhibiting weed growth in a field of transgenic crop plants comprising first planting the transgenic plants transformed with an expression cassette comprising an isolated polynucleotide molecule having gene regulatory activity and comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-7 and operably linked to a DNA molecule encoding a glyphosate tolerance gene and then applying glyphosate to the field at an application rate that inhibits the growth of weeds, wherein the growth and yield of the transgenic crop plant is not substantially affected by the glyphosate application. The glyphosate application rate is the effective rate necessary to control weeds in a particular glyphosate tolerant crop; these rates may range from 8 ounces/acre to 256 ounces/acre, preferably 16 ounces/acre to 128 ounces/acre, and more preferably 32 ounces/acre to 96 ounces/acre. The glyphosate is applied at least once during the growth of the glyphosate tolerant crop and may be applied 2, 3, or 4 times during the growth of the crop or more as necessary to control weeds in the field.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
Four actin promoters were isolated from rice and two actin promoters were cloned from maize. Genomic DNA was generated from corn (Zea mays) and rice (Oryza sativa) tissue using standard methods familiar to one skilled in the art. The genomic libraries were prepared according to manufacturer instructions (GenomeWalker™, CLONTECH Laboratories, Inc, Palo Alto, Calif.). In separate reactions, genomic DNA was subjected to restriction enzyme digestion overnight at 37� C. with the following blunt-end endonucleases: EcoRV, ScaI, DraI, PvuII, or StuI (CLONTECH Laboratories, Inc. Palo Alto, Calif.). The reaction mixtures were extracted with phenol:chloroform, ethanol precipitated, and resuspended in 10 mM Tris buffer pH 8.5. The purified blunt-ended genomic DNA fragments were then ligated to the GenomeWalker™ adaptors. Ligation of the resulting DNA fragments to adaptors was done according to manufacturer protocol. The GenomeWalker™ sublibraries were aliquoted and stored at −20� C.
Four rice actin genes were aligned to design a gene-specific primer capable of annealing to multiple actin gene sequences. Three corn actin genes were aligned to design a gene-specific primer capable of annealing to multiple actin genes. Genomic DNA from corn and rice ligated to the GenomeWalker™ adaptor was subjected to PCR amplification in separate reactions with either the rice or corn gene-specific primer and a primer designed to anneal to the GenomeWalker™ adaptor. Standard protocols provided by the manufacturer were used. Those of skill in the art are aware of the variations in PCR conditions including choice of polymerase, cycling conditions, and concentrations of the reaction components. PCR products were cloned into pUC19 and the DNA insert was sequenced for each clone. The presence of an actin gene sequence in the insert sequence was used to identify the upstream promoter region.
The isolated promoters were cloned into an appropriate plant expression vector for the subsequent characterization of the promoter in plants. The DNA fragments resulting from the nested PCR amplification described above were isolated and gel purified using methods familiar to those skilled in the art. The purified DNA was digested with one or more restriction endonuclease(s) to permit ligation into a suitable cloning or expression vector. The promoter fragments were incorporated into a plant expression vector by positioning the promoter fragments in linkage with a reporter gene by restriction enzyme digestion and ligation using methods well known in the art. The purified DNA of the present invention was ligated as a NotI/NcoI fragment into a vector containing the necessary plant expression elements, including the GUS or At-CP4-EPSPS transgene operably linked to the promoter. An aliquot of the ligation reaction was transformed into a suitable E. coli host such as DH10B and the cells were plated on selection medium (100 μg/ml spectinomycin). Bacterial transformants were selected, grown in liquid culture, and the plasmid DNA was isolated using a commercially available kit such as the Qiaprep Spin Microprep Kit (Qiagen Corp., Valencia, Calif.). Purified plasmid containing the predicted insert size was DNA sequenced in both directions using the dye terminator method and oligonucleotide primers designed to anneal to the region of the vector bordering the promoter insertion site. Additional oligonucleotide primers for further DNA sequencing of the promoter were then prepared based on the sequence produced from the results of the first DNA sequencing reaction. This was repeated until a full-length sequence of the isolated promoter was produced.
Corn protoplasts transformed with a vector containing a promoter operably liked to the GUS transgene were analyzed for GUS expression levels, measured by GUS activity as MU (pMol/ng of Total Protein). GUS expression levels of the actin promoters in protoplasts were compared to GUS expression levels in protoplasts transformed with a vector having the Os-actin 1 (ract1) promoter driving GUS and a vector having the Os-actin 2 promoter driving GUS. Protoplasts transformed with a vector containing only the luciferase transgene (LUC) were used as a negative control for GUS activity. Data are provided in Table 1.
GUS activity measurements in corn protoplasts
Promoter operably
linked to GUS
Seq ID Num
GUS activity
P-Os-actin 1
P-Os-actin 2
Corn protoplasts transformed with a vector containing a promoter operably linked to the At-CP4-EPSPS transgene were analyzed for CP4 expression levels, measured as CP4-EPSPS accumulation (ng of CP4/mg of total protein). CP4-EPSPS expression levels of the actin 5 promoters in protoplasts were compared to CP4-EPSPS expression levels in protoplasts transformed with a vector having the Os-actin 1 (ract1) promoter driving At-CP4-EPSPS and a vector having the e35S promoter driving At-CP4-EPSPS. Protoplasts transformed with a vector containing only the luciferase transgene (LUC) were used as a negative control for GUS activity. Data are provided in Table 2.
CP4-EPSPS accumulation measurements in corn protoplasts
Promoter operably linked to
Related Figure
CP4-EPSPS accumulation
P-Os-actin 1
P-Os-actin 2
Transgenic corn plants transformed with a vector containing a promoter operably linked to the CP4-EPSPS transgene were analyzed for transformation efficiency, copy number, and transgene expression level. Transformants were analyzed for transformation efficiency measured as the percent of explants produced (embryos that regenerated to form plants) compared to the total transformed. Genomic DNA from transgenic plants was used to determine the percent of single copy events out of the total number of transgenic plants analyzed. Single copy events were used to determine CP4-EPSPS transgene expression levels, measured as the percent of CP4-EPSPS accumulation relative to Roundup Ready� corn line NK603. CP4-EPSPS expression levels of the actin promoters were compared to CP4-EPSPS expression levels in transgenic plants transformed with a vector having the Os-actin 1 (ract1) promoter driving CP4-EPSPS and a vector having the e35S promoter driving CP4-EPSPS. Data are provided in Table 3.
Measurements of transgenic corn plants
operably linked
Single Copy
to CP4-EPSPS
Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications that are within the spirit and scope of the appended claims. All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.
|Cited Patent
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|Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthase, production and use
|Sep 4, 1992
|May 17, 1994
|Monsanto Company
|Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
|Feb 21, 1995
|Oct 31, 1995
|Monsanto Company
|Glyphosate tolerant plants
|Apr 26, 1994
|Feb 6, 1996
|Dekalb Genetics Corporation
|Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
|Jun 7, 1995
|May 6, 1997
|Monsanto Company
|Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
|Sep 13, 1994
|May 27, 1997
|Monsanto Company
|Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
|Oct 27, 1993
|Jun 24, 1997
|Cornell Research Foundation, Inc.
|Rice actin gene and promoter
|Apr 3, 1997
|Mar 21, 2000
|Dekalb Genetics Corporation
|Glyphosate resistant maize lines
|Oct 29, 2001
|May 1, 2003
|Maxygen, Inc.
|Novel glyphosate N-acetyl transferase (GAT) genes
|Sep 24, 2004
|Jul 20, 2006
|Stanislaw Flasinski
|Actin regulatory elements for use in plants
|Mar 23, 2001
|Jan 25, 2007
|Boukharov Andrey A
|Genomic plant sequences and uses thereof
|Jan 4, 1991
|Jul 11, 1991
|Cornell Research Foundation, Inc.
|Rice actin gene and promoter
|Apr 3, 1998
|Oct 8, 1998
|Dekalb Genetics Corporation
|Glyphosate resistant maize lines
|Oct 5, 1999
|Apr 13, 2000
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|Maize promoters
|May 12, 2000
|Nov 23, 2000
|Dekalb Genetics Corporation
|The rice actin 2 promoter and intron and methods for use thereof
|An et al., "Strong, constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues," The Plant Journal, 10:107-121, 1996.
|Database EMBL Accession No. AP003263, Feb. 22, 2001.
|Dolferus et al 1994, Plant Physiology 105:1075-1087, provided in Applicant IDS.
|Dolferus et al., "Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene," Plant Physiology, 105:1075-1087, 1994.
|Donald et al 1990, EMBO J. 9:1717-1726, provided in Applicant IDS.
|Donald et al., "Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter," EMBO J., 9:1717-1726, 1990.
|GenBank accession No. X15862.
|GenBank accession No. X15863.
|GenBank accession No. X15864.
|GenBank accession No. X15865.
|Kim et al 1994, Plant Molecular Biology 24:105-117, provided in Applicant IDS.
|Kim et al., "A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity," Plant Molecular Biology, 24:105-117, 1994.
|McElroy et al. Plant Cell 1990 vol. 2 pp. 163-171.
|McElroy et al., "Characterization of the rice (Oryza sativa) actin gene family," Plant Mol. Biol., 15:257-268, 1990.
|McElroy et al., "Construction of expression vectors based on the rice actin 1 (Act1) 5' region for use in monocot transformation," Mol. Gen. Genet., 231:150-160, 1991.
|McElroy et al., "Isolation of an efficient actin promoter for use in rice transformation," Plant Cell, 2:163-171, 1990.
|McElroy et al., "Structural characterization of a rice actin gene," Plant Mol. Biol., 14:163-171, 1990.
|McElroy et al., "Construction of expression vectors based on the rice actin 1 (Act1) 5′ region for use in monocot transformation," Mol. Gen. Genet., 231:150-160, 1991.
|Meagher, "Divergence and differential expression of actin gene families in higher plants," Int. Rev. Cytol., 125:139-163, 1991.
|Moniz de sa et al., "Phylogeny and substitution rates of angiosperm actin genes," Molecular Biology and Evolution, 13(9):1198-1212, 1996.
|Reece et al., "Genomic nucleotide sequence of four rice (Oryza sativa) actin genes," Plant Mol. Biol., 14:621-624, 1990.
|Reece, "The actin gene family of rice (Oryza sativa L)," Ph.D. thesis, Cornell University, Ithaca, NY, 1988.
|Wang et al 1992 Molecular and Cellular Biology 12:339-3406.
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|Zhang et al., "Analysis of rice Act1 5′ region activity in transgenic rice plants," Plant Cell, 3:1155-1165, 1991.
|U.S. Classification
|536/24.1, 435/320.1, 435/468, 800/278, 536/23.1, 800/295, 435/410
|International Classification
|Cooperative Classification
|European Classification


text:
It's hard to run a waste and recycling operation when you have to close down your landfill to minimize blowing debris every time it gets too windy. We worked with the "windy" City of Lethbridge to make its waste and recycling centre's operation and householder experience more debris-free and headache-free.
Our proposed design included modifications for one of the buildings to house a 45-ton-per-hour waste-baling-and-bagging facility which would compact waste (and stop the blowing debris). This has reduced the facility's required building footprint (it now takes up less space), while other site modifications such as a new entrance, dual-weigh-scale and scale-house facility, compost facility, and surface water management facility have reduced operating costs.
The experience of recycling and waste disposal was revamped to make it more straightforward. The transformed drop-off area now features clear signage identifying where recyclable items should go and better traffic management for enhanced user safety. Encouraging diversion of waste from landfill and more recycling visits, this leaner, greener, and more streamlined facility now remains open for business even on the windiest days.


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MAS Urges DOB To Re-Evaluate New Rule
March 6th, 2009, 11:15 am
Update via MAS Twitter: DOB Rule Breaking News: as MAS asked, DOB delays start-date until mid-April (instead of 3/9) for more public input twitter.com/MASNYC.
At a public hearing this afternoon, MAS will urge the Department of Buildings to further evaluate two new rules on the public challenge process for building approvals. The rules are scheduled to be implemented on Monday, March 9.
"While DOB's new rules are a commendable effort to advance public discourse on building approvals, MAS believes that further evaluation of the public challenge process and careful consideration of today's testimony should take place before the rule is put into effect," said David Schnakenberg, Menapace Legal Fellow for the Municipal Art Society.
MAS suggested that DOB convene a working group to consider the testimony and the input it has received today. MAS offered to participate in any way that might be helpful to improving the rule and realizing the Mayor Bloomberg's goal of meaningful public review of DOB approvals.
MAS also offered the following recommendations:
- While providing building diagrams in advance of construction is an improvement over existing practice, MAS does not feel that the information required of the applicant is sufficient, and asked that DOB expand its requirements to include a detailed zoning analysis.
- MAS urged DOB to improve its notification procedures, to extend the period in which the public may submit challenges, and to lessen the technical requirements incumbent upon challengers. Applicant postings are useless absent meaningful notice to the community and a genuine opportunity to challenge approvals.
- DOB must utilize available technology to better inform the public of approvals if it is to rely on improved public participation as a justification for shortening the challenge period.
- DOB must extend the public challenge period specified in the proposed rule. MAS recommended a minimum of 45 days, as opposed to 30, to allow for community board participation, as well as to allow for consultation with zoning or legal experts.
- MAS urged DOB to reconsider the requirement that the public must cite zoning and constructions and either strike it from the forms or explicitly limit the required citation to that which is on applicants' forms.
- To ensure that the challenge process is best utilized, MAS recommended that DOB establish an office in each borough, to assist individuals with public challenges.
- MAS also suggested that DOB dedicate resources to provide training in the use of the proposed challenged process.


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An Austrian first-time director Sunday defended his chilling Cannes entry about the relationship between a paedophile and his prey which shocked audiences and sharply divided critics.
The plot of "Michael", one of 20 contenders for the festival's top prize, has strong parallels to the story of Natascha Kampusch, the Austrian girl kept locked in a cellar for more than eight years by a child molester.
Film-maker Markus Schleinzer presents drab officer worker Michael, who keeps 10-year-old Wolfgang prisoner in a soundproofed cell behind a bolted door in his own basement, not as a monster but as a mild-mannered everyman.
"I wasn't interested in a witch hunt," said Schleinzer, 40, best known as a casting director on films by his compatriot Michael Haneke including the 2009 Palme d'Or winner "The White Ribbon".
"I wanted someone who seemed pretty average, where one could say, 'I know someone like that'. You find paedophilia, like all kinds of crime and violence, in every social stratum," he told AFP.
The film's casts a cold, clinical and riveted gaze on the perpetrator and observes the familiar routine that he and his young prisoner share.
Michael arrives at work each morning, performing well enough in his insurance job to earn a promotion, and even goes off on a ski holiday with friends, leaving Wolfgang in his custom-built prison with some instant soup.
The sexual abuse is mercifully shielded from view but the sight of a leering Michael reclining on Wolfgang's twin bed and calling the boy to him was enough to make viewers' blood run cold.
Wolfgang, an intelligent child who does not believe Michael's claim that his parents have abandoned him to his life in the dungeon, flinches every time his captor touches him.
But they also share relaxed moments together -- preparing dinner, doing jigsaw puzzles and decorating the Christmas tree. That down-time reveals a sentimental, even child-like side to Michael.
"Depicting criminals as monsters is in my opinion about protecting yourself and the desire to distance yourself from them," Schleinzer said. "If you make him a monster, what is he? A mythical creature? But he is not human."
Schleinzer said the Kampusch case was only one impetus for the story, noting that thousands of children vanish every year.
"Of course these issues are enormously frightening," he said. "But to suppress them for that reason and not talk about them does not minimise the danger."
The film sparked strong interest at Cannes, with an extra screening hastily arranged to accommodate critics who were shut out of the packed premiere.
Screen magazine called the picture "acutely sensitive to issues of exploitation" in a rave review.
"Michael could be your son, brother, father or colleague," its critic said.
"(And) the restraint of (Rauschenberger's) performance is in tune with a film which takes the sting out of a white-hot topic and transforms it into a troubling, thought-provoking and quietly disturbing drama."
Dennis Lim of the New York Times also gave the movie a strong endorsement when polled for Sunday's film trade journals.
But other critics such as Stephane Delorme at Cahiers du Cinema and David Stratton of The Australian gave the picture a "poor" rating, and Entertainment Weekly reviewer Lisa Schwarzbaum said it gave her "deep queasiness".
"What purpose does this movie serve? And why did Festival programmers choose to include this self-absorbed exercise among the precious competition films aside from, you know, shock value?" she wrote.


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Information about State Fair Community College evening programs, and registering for discussion sections and department seminars. Which college degrees give you the best chances of entering a successful career path after graduation? Concentrating on State Fair Community College evening programs, for example, may give you additional focus in the admissions selection process. Check with the admissions office for assistance with prerequisites, course registration deadlines, and college degree program requirements. Please use the form above to request admissions info for State Fair Community College.
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The Cult Trailer (2012)
01 June 2012 140 mins
Enter this dark world of sexual awakening. Where pain and pleasure go hand in hand. Devilishly rampant robed men are dominated by beautiful glamorous women. Give in to your darkest sexual urges and join Gazzman's The Cult Only a woman can truly make a woman submit. We learn this in this hot and steamy lesbian scene with British Babe Megan Coxxx, black stunner Skin Diamond and voluptuous Valentina. Hard girl girl girl action at its best. Curvaceous beauty Linet Slag lives up to her name in this ass stretching and pussy pounding double penetration scene that will leave you breathless. Tantalizing stuff! Dark haired beauty Valentina gives into her darkest desires. Masturbating just isn't enough for this hussy as she soon needs her ass fucked hard by a cloaked muscular stud.
Watch the official The Cult 2012 trailer in HD below.


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Lisa O'Connor/ZUMAPress.com
Lisa O'Connor/ZUMAPress.com
A hurricane isn't just a storm, an eclipse isn't just a dark day, and Megan Fox isn't just a hot star who says goofy things.
Some phenomena just require their own descriptive term.
So there oughta be a word for that. And that's where you and Celebrity Addictionary come in. Create a word to describe her unique qualities, her sublime totality, her utter Megan Foxness. Put it in the widget.
We need it. The language needs it. Megan Fox needs—no, deserves—it.
So make it a good one.


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Apprenticeship opportunities were celebrated in Scarborough with a week marking the benefits of 'earn while you learn' programmes.
National Apprenticeship Week saw a number of events across the town, including an apprenticeship fair organised by Scarborough's Jobcentre Plus.
Held at the Town Hall, the event brought together nine organisations and showcased more than 40 different apprenticeships on offer.
Scarborough MP Robert Goodwill has also spent time in the town looking at how apprentices contribute to the economy.
He visited The Academy in Roscoe Street, where he talked to hair and beauty apprentices and found out how they studied at the new centre in Scarborough while working at salons across the town.
He also enjoyed a haircut and manicure by the students while he was there.
Mr Goodwill also visited YH Training Services in Falsgrave Road to praise local learners and employers for their dedication to skills qualification and training. He spent time with each apprentice and discussed what qualification they were undertaking, and also who they were employed with.
Mr Goodwill then gave a talk about the future of Apprenticeships, the local economy and the struggle for young people to get into employment.
While in Scarborough Mr Goodwill also visited Yorkshire Coast Enterprise in Northway; a business start up support and development agency in Scarborough, Whitby and Ryedale. He learnt how the organisation supports entrepreneurs to help get their business venture off the ground, with a view to assisting the local economy.
Mr Goodwill said: "Yorkshire Coast Enterprise is helping hundreds of aspiring entrepreneurs get the finance and support to strike out on their own and launch their own business."


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Recovering Grace: A Gothard Generation Shines Light on the Teachings of IBLP and ATI
Labels: performance spirituality, resources
3 comments:
AnonymousJuly 23, 2011 at 9:18 PM
can anyone tell me how IBLP and ATI differ from IFB?
Not by much in practice, anonymous.
IFB are churches. IBLP/ATI is a parachurch organization. But the rules/principles/ideas are not too much different in a lot of ways.
Someone else can probably answer better than I can, though.
As someone who grew up IFB, your description is pretty much spot on, Sharon.
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Gerhart hoping to be more of a factor for Vikings in 2011
By Jon Krawczynski
AP Sports Writer
EDEN PRAIRIE -- Every time Toby Gerhart touched the ball in his first preseason with the Minnesota Vikings last year, he was hit and hit hard.
And that was just in practice against his teammates.
The golden boy from Stanford was given a rude welcome to the NFL in his first training camp, getting slammed to the turf by the likes of Pat Williams and Ray Edwards any chance they got.
"At the time, I was like God, everybody's out for my number," Gerhart recalled with a chuckle. "No matter what I did, I was getting hit. But it was all good, you get hit as a running back, and it was expected as a rookie. Can't complain, I guess."
Vikings defenders always seemed to put a little more mustard on their hits when No. 32 had the ball, and the message was clear -- Gerhart wasn't in Palo Alto anymore.
After leading the nation in rushing and touchdowns for the Cardinal, the Vikings traded up in the second round to grab him as a complement to Adrian Peterson. But Gerhart had a quiet rookie season, rushing just 81 times for 322 yards and a touchdown in 15 games.
It wasn't the kind of impact he expected to have after such a decorated college career.
"I was a little hesitant, a step slow, kind of thinking too much," Gerhart said. "This year I'm just relaxing, playing, doing what I always did. I feel much more confident and relaxed, and I think this year I can contribute in a lot of different ways."
It's no secret who runs the show in the Vikings backfield. Peterson is the first, second and third option on offense, one of the most dynamic players in the NFL and a guy who wants the ball in his hands as much as possible.
The offense new coordinator Bill Musgrave is installing will revolve around Peterson, so there is still some question about how much opportunity there will be for Gerhart to get in a rhythm and get rolling like he was used to doing in college.
Even with Peterson only playing just a few plays in the preseason opener in Tennessee last weekend, Gerhart had just three carries for 8 yards. The sparse workload has taken some getting used to for a workhorse who carried the ball 553 times in his final two years at Stanford.
Only three times last year did he get more than five carries in a game, including 22 touches for 76 yards and a score against Washington.
"It's a bit different," he said. "As a running back, it's hard to get that rhythm when you carry one carry and a quarter goes by and you get another carry, but you've got to make the most of it when you get it. For me last year it was pass protection, running routes like that, fine tuning those and contributing in any way possible."
With the lockout wiping out the summer, Musgrave is getting his first up-close look at Gerhart this preseason. He said Gerhart was "very sharp" and is picking up the intricacies of pass protection quickly.
"We know that Adrian and Toby will both complement each other was we go through the season," Musgrave said. "Two different running styles. We want to keep both of them healthy. It's a tough, tough way to make a living for those guys."
Also in the mix is Lorenzo Booker, an accomplished return man who has shown a little more wiggle and juice than Gerhart early this season.
"He's got skills that neither one of those two have in terms of catching the ball, splitting out of the backfield and having some receiver skills in his hopper," Musgrave said.
Left guard Steve Hutchinson said he already has noticed a change in Gerhart.
"Anytime you get a year under your belt and get through your rookie year, you're going to have a little more confidence in everything you do," Hutchinson said. "I think that's a big key to our offense is having a complement back there with Adrian."
So far in this camp, Gerhart hasn't taken near the amount of abuse he did as a rookie. Edwards and Williams are both gone, but the way Gerhart handled the treatment -- keeping his mouth shut and standing up for himself when he got into a little skirmish with Edwards -- seems to have resonated with his teammates and earned him some respect.
"Some of the guys, later on they said they just wanted to see what I had," Gerhart said. "It was kind of a test-the-waters sort of thing because they knew I'd be a guy that was gonna be with them down the road and they wanted to see what kind of guy I was. It was a learning experience to say the least."
NOTES: WR Percy Harvin watched much of practice after leaving with an apparent rib injury. He told reporters he was OK when practice was over. ... RG Anthony Herrera was back in his starting spot for 11-on-11 drills while he continues to work his way back from knee surgery. Musgrave said it was too early to tell if he could play on Saturday against Seattle. "So far, so good, first couple days he's been out here," Musgrave said. ... TE Visanthe Shiancoe (hamstring), LB Heath Farwell (hamstring), LB Kenny Onatolu (foot), DE D'Aundre Reed (calf) and CB Simeon Castille (hamstring) all sat out.


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Bossier City Police Department:
BOSSIER CITY, La. (KYTX) - The Bossier City Police Department is asking for the public's help in locating a runaway teenager. Tyrecia King, 15, of Bossier City was last seen on February 20, 2013 after she left home for school. Detectives have classified her as a runaway and have found nothing to indicate foul play being involved. However, they along with Tyrecia's family are concerned for her welfare.
Tyrecia is a black female, 5'9" tall, and weighs 280 pounds. Anyone who has any information regarding her whereabouts is urged to call Bossier City Police at (318) 741-8605.


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Business TypeTrading Company
Main Markets
Product/ServiceTrade Of Apparel & Fashion Category
Business Owner
Employees5 - 10 People
Link ToolIndonesia Apparel - Indonesia T-Shirts
Company TagsApparel - Garment Enquiry - Garment Stock Lots - Indonesia - Jakartaindonesia
Company Introduction
We are garment trading & agent located in Jakarta-Indonesia. We can also offer garment stock lots. We can provide your garment enquiry in big amount and competitive price for good quality. Also will arrange the shipment as well.
Contact Information
Contact PersonMs. Elga R
Fax Number62-21-88322220
Post Code13930
AddressCatelaya C5/35, Bekasi, Propinsi Jawa Barat, Indonesia
Online Mapelgacollection Map & Directions


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When an individual borrows money from a bank or a financial institution in return for a guarantee, it is a loan. Check out more about loans by scrolling down.
A Credit Union Vs. Bank Comparison: Which Should You Choose?
Wondering who will win the age-old financial debate of credit unions vs. banks? This Buzzle post will tell you about the pros and cons of both, credit unions and banks, which will help you make the right investment decision.
Hardship Loans
An economic crisis or upheaval affects everyone, and many of them have to avail hardship loans to overcome their financial troubles or repay off their debts.
Pawning Vs. Selling
There is a huge difference between pawning and selling a item. The pawning vs. selling comparison is highlighted in the article below.
How to Choose Between a Mortgage Broker and Bank
A mortgage broker and a bank are both institutions, which help you borrow loans for your requirements. This Buzzle article will help you understand how to choose between a mortgage broker and bank.
Things to Consider Before Applying for a VA Loan
Due to the nature of their work, people from the military go through distinct financial troubles. Their constant traveling compels other members in their family to keep up a steady employment, which is why this Buzzle article...
Loan Agreement Template
A basic know-how about drafting a loan agreement would prove beneficial for both lenders as well as borrowers. Here is a loan agreement template for your reference.
Line of Credit Vs. Loan
What are the exact differences between a line of credit and a loan? Read to know the distinction.
Loan Underwriting Process
Getting a loan approved is certainly a difficult task due to the hurdle of loan underwriting. The process of underwriting a loan can be quite lengthy because of the need to collective vital information of the borrower and ascertain...
Cosigner Responsibilities
One of the most important cosigner responsibilities is to make sure about the background and financial paying capability of the person who is looking forward to take a loan...
APR Vs. APY
Knowing the difference between APR and APY is useful in many ways while borrowing or saving money in banks. Read the article which discusses the distinction between them in detail, to help you understand the importance of each of...
Borrowing Against 401(k)
Borrowing against 401(k) for making a down payment on a house should be considered only after exhausting all other avenues of procuring loans. This article talks about the pros and cons of borrowing against 401(k).
Private Money Lending
Private money lending is a boon to the borrowers, who are unable to procure money from banks and credit unions.
What are Residential Hard Money Loans
Residential hard money loans fill the gap left by institutional financing, which has become scarce due to economic instability. These money lenders provide loans which are related to real estate properties, at high interest rates,...
What is a Subprime Loan
A way of making easy money, the subprime loans have been debated since they were originated. It was rightly said that easy money is bad money.
How to get Small Loans and Grants for Women
Getting small loans and grants as a woman entrepreneur is not a difficult task at all. Millions of dollars are set aside by the government as grants, and these can be claimed by women aspiring to be business owners.
Sources of Consumer Credit
Credit plays a vital role in everyone's life, in some form or the other. Read on for some important sources of consumer credit.
What is an Auto Title Loan
An auto title loan is a short-term loan that is secured by the title of your car. Read the following article that will help acquaint you with some important aspects related to it.


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15 € voucher15€ Off orders exceeding at least 300€
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With the Braun stick Blender TributeCollection MQ 100 soup can you conjure up the favorite mash of your treasure. The hand blender is very easy and is easy to use.
The extremely hard stainless steel knife blades guarantee the uniform and fine Pürier results. A backsplash provides a clean and pleasant work.
The Braun stick Blender TributeCollection MQ 100 soup has a speed and consists of a high quality plastic housing. A transparent mix and measuring cup is included. It has a capacity of 600 ml.
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Silicone Oven Glove
Please follow and like us:
Silicone Oven Glove We are a professional china silicone mold and molding company, an audited supplier by SGS, mainly engaged in silicone products, including household items and promotional gifts, such as bakeware& kitcheware, baking gloves, steamers & bowls, baby feeding bottles, wristbands, ashtrays, etc… 1. 100% food-grade(FDA, SGS, LFGB) silicone material 2. Size: 212*161*37mm 3. Weight: 112g 4. Non-stick finish 5. Temperature range: -40 to 230 degrees Celsius 6. Flexible and easy to clean 7. Any colors are available. if you want to make silicone product or looking for a professinoal china silicone rubber supplier as your partner,please feel free to contact us.


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Gorakhpur, Mar 8: Reproaching the Congress led-United Progressive Alliance (UPA) regime at the Centre, Bharatiya Janata Party (BJP) today said the former's mistakes would prove beneficial to the party to form a government at the Centre.
''All promises of the UPA government have fallen like a pack of cards. Masses have come to know about the anti-people face behind it.
Common man is looking for a change and BJP PM nominee Lal Krishna Advani is the only choice left,'' UP BJP chief Ramapati Ram Tripathi said addressing the party's two-day executive meet here.
''Dr Manmohan Singh will always be the weakest Prime Minister...owing to the Centre's wrong policies, prices of essential commodities are sky-rocketting. It has lost control on the market. Therefore, the petrol and diesel prices are persistently showing an upswing trend,'' he underlined.
However, only these mistakes would prove favourable to BJP to form a government at the Centre, Mr Tripathi maintained.


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I did my first work with webos 2.2.4 on my pre3.
If you want to view pdf view in landscape mode. try this guide:
Use Webos Quick install to Sent the file (attacked) (Tool/sent file) to the location:
Restart and you can rotate the pdf view, read it on landscape mode.
But there is one broblem! I can't read all the page when pdf view in landscape mode... Need more help!
Thank for any input information!
If you want to lock and unlock the rotation pdf view and another apps use this guide: xwTweak 0.1.0 - Global Accelerometer Switch
Update 1:
More useful patch for pdf view I've known:
1. Hide Header patch: [PATCH] Hide Annoying Header In PDFViewer (tarirene) (useful in the landscape mode)
2. Hide Page count patch by tarirene: [PATCH REQUEST] PDF View Improvements
It's better for lager view on Pdf view.
Update 2:
And for who installed both patches: Pdf view will not display full screen (there is a black bar at the bottom). You can sold this problem by using the document-assistant2.zip


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Manchester law firm Donns has played a part in the first successful action for compensation brought by a British soldier against the US military.
The case, which took place in Atlanta, saw a Donns partner, under the unusual title of English Co-Counsel, sitting on the counsel bench.
Partner Hilary Meredith was acting on behalf of the Whitley family from York, whose son, a Lieutenant in the British Army, was killed in 1993, in a coach accident in Atlanta.
The coach and driver were supplied by the US Army, and it was later discovered that the driver had been drinking and had a record of drug offences.
The plaintiffs convinced the court that the British officers on the bus were there in a wholly civilian capacity. They were in Atlanta as part of a rugby tour.
In the US, no compensation is given to military personnel for injuries sustained while on duty.
Meredith said: "We are overjoyed with the result, and we can now press ahead with claims for the other 15 men who were injured on the same bus."