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Item #19030
Streamlined details and modern appeal characterize our Barnsley Desk Lamp. The silver-finish adjustable arm is supported by a matching vertical pole rising from a tapered, bi-level platform in black. The black-finish banker-style shade features an open construction that shows off the included antique-style light bulb. Position this task light however you wish by maneuvering the black-finish handle.
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text: What a Beautiful MorningIt was a sunny morning. Sunlight streamed through the trees and was welcomed by all the Clan members, some of them basking in the pockets of light. Many of them shared tongues, shared news of the day, chatting excitedly about training or the warm weather. The Clan seemed to be recovering well from the fox attacks.Breezepaw was in Patchstar's den. Having delivered kits before, the medicine cat was calm, and Patchstar matched his temperament. It wasn't her first litter either; the kits were born easily with no problems. Breezepaw congratulated Patchstar before fetching Dave from the Elders' den. Breezepaw put his tail on Dave's shoulder and the young retired warrior stepped eagerly into the leader's den before laying next to Patchstar. His scars were still prominent but most had already healed, at least to a degree; nevertheless, the tortoiseshell leader couldn't help but purr when she saw him."Dave...we have six kittens." Patchstar purred excitedly."Six..." Dave repeated almost breat


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Lixit prepares for growthJuly 21, 2014
NAPA — Lixit Corp., which has captured more than two-thirds of the U.S. market for small-pet watering devices, is preparing to reel in more dog accessories sales with the acquisition of a Chicago-area pet products company that patented a reel-based dog run, and a new Napa warehouse is planned to support Lixit's double-digit annual growth.
Demand rises as SBA refi program endsSeptember 10, 2012
North Bay lenders are lamenting the imminent expiration of the U.S. Small Business Administration's temporary 504 refinance program, which allows a one-time refinance of debt and reinvestment of equity.
The temporary program, launched by the Jumpstart Our Small Businesses (JOBS) Act of 2010, allows a small-business owner to refinance debt through the typical 504 loan structure used for financing property and other tangible assets: a 10 percent equity contribution from the borrower, a 40 percent, 20-year fixed-rate government loan and a 50 percent second mortgage from a second commercial lender. In combination, up to $10 million can be refinanced through the program.
CFO Recognition Awards 2012: Laurie Corona, Lixit Corp.August 13, 2012
Executive vice president and chief financial officer, Lixit Corporation, Napa. Financial leaders in the current economy should work to "relay financial information to other leaders in the organization so that sound business decisions can be made."
Manufacturing Awards 2012: Community: Lixit Corp.July 16, 2012
NAPA — Lixit Corporation, as its name implies, is the largest small animal watering device manufacturer in the world, with a 70 percent market share in its category and revenues in excess of $10 million annually. The firm has been growing at a double-digit rate for the past couple of years, despite a five percent decline during the recession.
Business Journal announces manufacturing awards winnersJune 13, 2012
The North Bay Business Journal is proud to present the 2012 Top Manufacturers Awards to 13 North Bay companies.
"We want to recognize the contribution all North Bay manufacturers add to our local economy, through high-wage jobs, training and exports that bring critical dollars back into our economy and communities," said Business Journal Editor and Associate Publisher Brad Bollinger. "They are terrific employers and members of the community. And they bring innovation to our region in a big way."
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Additional file 6: Figure S.
Lamp1-GFP partially colocalizes with LTR. Colocalization of the reporter Lamp1-GFP that labels primary lysosomes, late endosomes and digesting lysosomes is not complete with LTR, a dye that stains acidic structures only. Boxed area is shown enlarged. Scale bar equals 20 μm.
Format: PDF Size: 151KB Download file
This file can be viewed with: Adobe Acrobat Reader
Lőw et al. BMC Cell Biology 2013 14:29 doi:10.1186/1471-2121-14-29


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Town Square
Paly grad Whitson earns player of week volleyball honors
Original post made on Sep 29, 2011
Read the full story here Web Link posted Thursday, September 29, 2011, 9:00 AM
Comments (1)
a resident of Palo Alto High School
on Sep 29, 2011 at 4:15 pm
I am appalled with the Palo Alto Online's decision to compare the Whitson sisters in this story. Both sisters are incredibly talented, and it is completely unnecessary to claim that "age has the edge" when both sisters stand out in their respective age groups. The two sisters also play different positions which require different skill sets, making it impossible to compare the two accurately. Why would a community newspaper ever insinuate that there exists a competition within an extremely supportive family? This article is unnecessary and offensive; instead of reflecting well on either of the Whitson's, it reflects very poorly on the Palo Alto Online.
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Grab a Bowl of Heaven soon in Mountain View
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text:
A suspicious object at a business premises sparked the third alert on Thursday evening.
Sloan Street was closed while army bomb experts examined a holdall reported by a concerned member of the public. It was found not to contain anything untoward.
Largymore Primary School was also evacuated in an earlier security alert.
Army technical officers carried out a number of controlled explosions on items in a stolen car - but these were subsequently determined to be not suspicious.
Mercer Street, between the Hillhall Road and Saintfield Road has now reopened.
Meanwhile Barrack Street in Lisburn reopened following another operation at Lisburn Police Station.
The incident happened shortly before midday, when a suspicious object was brought to the station by an unsuspecting member of the public.
A PSNI spokesperson said the object was examined and seized for investigation.
Police would like to thank residents and motorists for their support and patience throughout the security operations in Lisburn today.— PSNI (@PoliceServiceNI) May 29, 2014
Police would like to thank residents and motorists for their support and patience throughout the security operations in Lisburn today.
— PSNI (@PoliceServiceNI) May 29, 2014
Woman dragged from car in city hijacking
Man charged over city AK47 find
Robinson warns over NI's finances
Masked man robs service station
Man released over £70k cannabis find
Man remanded over fight outside bar
Man assaulted during burglary
Only The Young in shock X Factor exit
Shot fired at house in Derry
Citi announces 600 new jobs
AK47 arrest linked to grenade attack
Ardoyne bandsmen accused of breach
Passengers hit in Belfast bus attacks
Ulster Bank to close 10 branches
Safety call following child's death
Ulster Bank parent hit with £56m fine
Man cautioned for courts' graffiti
Lee Evans retiring at the age of 50


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We created Pandora to put the Music Genome Project directly in your hands
It's a new kind of radio –
stations that play only music you like
Aydan the turtlegirl
- Hometown: Gastonia NC
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text: Show StoriesRSS select from Issues all issues Democrats EconomyCorporations Energy Jobs Poverty Small Business Taxes Unions Wall Street EducationEarly Education School Reform ElectionsElection 2012 Election 2014 Election 2016 Money in Politics Voting Rights EqualityImmigration LGBT Race Women GreenClimate Pollution HealthFood Global Health Health Care Reform HIV-AIDS Sexual Health National SecurityAfghanistan Middle East Military Terrorism RepublicansTea Party SocietyCivil Liberties Criminal Justice Culture Guns Media Religion all issuesDemocratsEconomy- Corporations- Energy- Jobs- Poverty- Small Business- Taxes- Unions- Wall StreetEducation- Early Education- School ReformElections- Election 2012- Election 2014- Election 2016- Money in Politics- Voting RightsEquality- Immigration- LGBT- Race- WomenGreen- Climate- PollutionHealth- Food- Global Health- Health Care Reform- HIV-AIDS- Sexual HealthNational Security- Afghanistan- Middle East- Military- TerrorismRepublicans- Tea PartySociety- Civil Liberties- Criminal Justice- Culture- Guns- Media- Religion any time Past hour Past 24 hours Past week Past month By date any timePast hourPast 24 hoursPast weekPast monthBy date Start date Date E.g., 11/24/2014 End date Date E.g., 11/24/2014 An A+ for Jackie Robinson West 11/07/14 08:00PM Rev Al Sharpton gives out his report card. Little League sensations from Chicago managed to score top honors. Find out why and who else made the cut. watch Inside the White House 'power lunch' 11/07/14 07:49PM The White House is slamming a new attempt to gut the Affordable Care Act, but with the Supreme Court announcing it will rule on a related lawsuit, is a compromise between parties possible? Rev. Sharpton talks to Joan Walsh and EJ Dionne. watch George Clinton: Redefining funk for a new generation 11/07/14 06:51PM Funk legend George Clinton stops by PoliticsNation to talk with Rev. Al Sharpton about his new book, "Brothas Be, Yo Like George, Ain't That Funkin' Kinda Hard On You?" watch Jerry Seinfeld sparks autism conversation 11/07/14 06:46PM Rev. Sharpton talks to Zerlina Maxwell, Chris Witherspoon and Caroline Modarressy-Tehrani about Jerry Seinfeld's latest interview with NBC News' Brian Williams and his comments about autism. watch Is 'feminist' now a dirty word? 11/07/14 06:42PM Rev. Sharpton talks to Zerlina Maxwell, Chris Witherspoon and Caroline Modarressy-Tehrani about a series of Hollywood women rejecting the title of feminist. watch Can Dems reach a compromise with the GOP? 11/07/14 06:40PM Rev. Sharpton talks to Zerlina Maxwell, Chris Witherspoon and Caroline Modarressy-Tehrani about the chances of democrats striking a compromise with leaders of the GOP who are currently trying to strip "Obamacare." watch Who really killed Osama bin Laden? 11/07/14 06:30PM Rev. Al Sharpton talks with Col. Jack Jacobs about the latest controversy with the SEAL Team Six members who killed Osama bin Laden and what's at stake. watch The GOP's gloomy outlook on the economy 11/07/14 06:16PM Rev. Sharpton talks with Jason Johnson about GOP outlook on the economy. The private sector has added jobs for 56 straight months – the longest streak on record. Still, Republicans say the president is holding up the economy. watch President Obama to nominate Loretta Lynch as Attorney General 11/07/14 06:14PM Rev. Al Sharpton reports breaking news that President Obama will nominate U.S. Attorney Loretta Lynch to be the next Attorney General of the United States. watch Rev's Early Reads: November 7 By Dann McDorman 11/07/14 07:00AM Who really killed Osama bin Laden?... Pres. Obama meets with McConnell, Boehner at White House... Rush Limbaugh praises gridlock. read more FL mayor responds to detaining 90-year-old volunteer 11/06/14 06:59PM I appreciate the opportunity to clarify much of the misinformation that has been prevalent in the media recently regarding the homeless. Contrary to reports, the City of Fort Lauderdale is not banning groups from feeding the homeless. We have established an outdoor food distribution ordinance to ensure the health, safety and welfare of our community. read more POTUS has comeback for Michael Jordan 11/06/14 06:55PM President Obama had a great comeback for Michael Jordan after His Airness talked down about Obama's golf game. watch Bin Laden's shooter wants credit for kill 11/06/14 06:50PM The Navy SEAL who shot Osama Bin Laden has allegedly revealed his identity so that he can profit from his role in the raid. Should he? Rev. Al talks to Liz Plank, Josh Zepps and Angela Rye for answers. watch GOP thanks Dems for keeping Obama on the bench 11/06/14 06:44PM Today members of the GOP poked fun at democrats that backed away from aligning themselves with President Obama before midterms. watch 90-year-old detained for feeding the homeless 11/06/14 06:32PM Rev. Sharpton talks to 90-year-old Arnold Abbot and Father Mark Sims who were detained in Ft. Lauderdale for feeding the homeless because of a controversial new ordinance. watch Pages< 1 … 3 4 5 6 7 8 9 10 11 … 363 >


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A series of independent non-transgenic mutations found in the fruit PG gene of tomato; tomato plants having these mutations in their fruit PG gene; and a method of creating and identifying similar and/or additional mutations in the PG gene by screening pooled and/or individual tomato plants. The tomato plants of the present invention exhibit reduced PG enzyme activity and fruit that soften more slowly post harvest without having the inclusion of foreign nucleic acids in their genomes.
1. A mutated tomato plant that produces a tomato fruit comprising at least one fruit polygalacturonase gene, wherein:
a) the tomato fruit has reduced fruit polygalacturonase enzyme activity that is less than 40% of the fruit polygalacturonase enzyme activity of a tomato fruit produced by a non-mutated wild type tomato plant;
b) the reduced fruit polygalacturonase enzyme activity is caused by a human-induced non-transgenic mutation in the fruit polygalacturonase gene of the tomato fruit of the mutated tomato plant, wherein the mutation correlates with a change from glycine to arginine at amino acid 178 of SEQ ID NO: 2; and
c) the tomato fruit of the mutated tomato plant softens more slowly than the tomato fruit produced by the non-mutated wild type tomato plant.
2. A tomato fruit of the mutated tomato plant of
3. Seeds or plant parts of the mutated tomato plant of
4. Seed resulting from a cross between the mutated tomato plant of
5. Pollen from the mutated tomato plant of
6. The mutated tomato plant of
This is a divisional of U.S. patent application Ser. No. 11/246,793, filed Oct. 7, 2005, which is a divisional of U.S. patent application Ser. No. 10/691,374, filed Oct. 22, 2003, which claims the benefit of U.S. Provisional Patent Application No. 60/420,228, filed Oct. 22, 2002.
This invention concerns mutations in the fruit polygalacturonase (PG) gene of tomato. This invention further concerns tomato plants having mutations in their PG genes. This invention further concerns a method that utilizes non-transgenic means to create tomato plants having mutations in their PG genes.
United States consumers spend more than $4 billion each year on fresh market tomatoes. During the summer months, most of these fresh market tomatoes are grown on farms located throughout the United States and then sold locally. During the cooler months, when locally grown tomatoes are not available, most of these tomatoes are grown in the southern portions of the United States and in Mexico and then shipped by truck throughout the rest of the country. Unfortunately, when these southern grown tomatoes are allowed to fully ripen on the vine before shipping, they do not remain in marketable condition long enough for supermarkets to shelve them and for consumers to buy them.
To prevent the tomatoes from rotting before they reach consumers, farmers typically pick, pack, and ship the tomatoes while green. Before sale, the green tomatoes are gassed with ethylene to redden them. These unripened ōgassedö tomatoes do not spoil quickly, but they have developed a reputation for poor flavor, especially compared to the summer ōvine-ripenedö tomatoes.
Due to consumer dissatisfaction with the unripened ōgassedö tomatoes, research and breeding efforts have focused on developing tomatoes that exhibit a longer shelf-life when they are allowed to ripen fully on the vine. One approach to developing longer shelf-life tomatoes is to use traditional breeding techniques, i.e., crossing tomato plants with desired characteristics and selecting those progeny plants with fruits exhibiting longer shelf-lives. While traditional breeding techniques have been used to develop most of the tomato cultivars used by growers today, these methods are very time intensive. It can take years to breed a novel tomato variety that may exhibit only a modest increase in shelf life.
Another approach to developing longer shelf-life tomatoes is to use genetic techniques to manipulate the biochemical and physiological changes associated with the ripening process in tomatoes. One biochemical change in ripening fruit is the depolymerization and solubilization of cell wall polyuronides by the ripening-induced cell wall degrading enzyme, polygalacturonase (PG). Tomato fruit PG (Della Penna et al., Proc. Natl. Acad. Sci. U.S.A. 1986 83:6420-6424; Bird et al., Plant Mol. Biol. 1988 11:651-662) belongs to a family of tomato PG genes. PG enzyme activity increases dramatically during the ripening of many fruits, including tomato, and is the primary enzymatic activity responsible for cell wall polyuronide degradation.
For example, in U.S. Pat. Nos. 5,107,065; 5,442,052; 5,453,566; 5,569,831; and 5,759,829, tomato plants were transformed with DNA constructs encoding an antisense oligonucleotide for the PG gene. When expressed, the foreign DNA provided an RNA sequence capable of binding to the naturally existing mRNAs of the PG gene in the transformed tomato plant thereby preventing the translation of the mRNA into the PG protein. The fruit of transformed tomato plants showed improved properties in terms of slower softening post harvest, thereby increasing the shelf-life of the tomato.
Another research group, using a complicated series of transgenic manipulations involving transposon sequences from another plant species, created a ōknock outö of the PG gene in tomato. Enzymatic analysis of fruit from plants containing the knock out of the PG gene showed at least a 1000-fold reduction in PG levels. See Cooley, M. B. and Yoder, J. I., Plant Mol. Biol., 1998 Nov. 1, 38(4):521-30; Cooley et al., Mot. Gen. Genet. 1996 Aug. 27, 252(1-2):184-194.
This anti-sense and ōknock-outö work indicates that fruit PG gene expression is not necessary for viable, normal tomato fruit production. While several features of the ripening process remain normal, transgenic tomatoes having reduced PG gene expression exhibit slower softening post harvest and increased shelf life. Additionally, these transgenic tomatoes exhibit a lower incidence of post-harvest disease infection due to the preservation of intact fruit skin and coat caused by the delayed softening. Therefore, the tomatoes with reduced PG have fewer cosmetic blemishes which deter customers.
Reduced PG enzyme activity is important not only to the fresh market tomato industry but also to the processed tomato industry. During commercial processing of tomatoes, pectin integrity of the tomato is lost by enzymatic degradation of the pectin by PG. In order to avoid this degradation, a rapid, high heat treatment is used to destroy the PG enzyme activity. The annual cost associated with the total energy required to bring millions of tons of tomatoes to a temperature sufficient to rapidly inactivate the PG enzyme is a significant cost to the tomato processing industries.
While the use of these genetic techniques has resulted in producing tomatoes with reduced PG gene expression, the genetic techniques used to date employ recombinant DNA being introduced into tomatoes. Since many consumers have clear preferences against genetically modified foods, it would be useful to have a tomato exhibiting reduced levels of fruit PG that was not the result of genetic engineering methods. However, to date, no one has ever found or described a naturally occurring ōknockoutö of the endogenous tomato PG gene. Therefore, a tomato with its fruit PG gene either knocked out or otherwise hindered would have tremendous value to the entire tomato industry.
In one aspect, this invention includes a tomato plant, tomato fruits, seeds, plant parts, and progeny thereof having reduced fruit polygalacturonase enzyme activity compared to the wild type tomato plants wherein the reduced fruit polygalacturonase enzyme activity is caused by non-transgenic mutation in the tomato fruit polygalacturonase gene.
In another aspect, this invention includes a tomato plant having tomato fruits which soften slower post harvest compared to wild type tomato fruits due to an altered polygalacturonase enzyme, as well as fruit, seeds, pollen, plant parts and progeny of that plant.
In another aspect, this invention includes food and food products incorporating tomato fruit having reduced polygalacturonase enzyme activity caused by a non-transgenic mutation in the fruit polygalacturonase gene.
In another aspect, this invention includes a tomato plant having reduced fruit polygalacturonase enzyme activity compared to the wild type tomato plants created by the steps of obtaining plant material from a parent tomato plant, inducing at least one mutation in at least one copy of a fruit polygalacturonase gene of the plant material by treating the plant material with a mutagen to create mutagenized plant material, culturing the mutagenized plant material to produce progeny tomato plants, analyzing progeny tomato plants to detect at least one mutation in at least one copy of a fruit polygalacturonase gene, selecting progeny tomato plants that have reduced fruit polygalacturonase enzyme activity compared to the parent tomato plant; and repeating the cycle of culturing the progeny tomato plants to produce additional progeny plants having reduced fruit polygalacturonase enzyme activity.
SEQ. ID. NO.: 1 shows the DNA sequence between the start and stop codons for the coding region of Polygalacturonase (Gen Bank Accession NO. M37304).
SEQ. ID. NO.: 2 shows the protein sequence encoded by SEQ. ID. NO.: 1.
SEQ. ID. NOS.: 3-46 show the DNA sequences for Polygalacturonase specific primers of the present invention.
SEQ. ID. NO.: 47 shows the DNA sequence of the Polygalacturonase gene for Mutation 13345.
SEQ. ID. NO.: 48 shows the protein sequence encoded by SEQ. ID. NO.: 47.
SEQ. ID. NO.: 49 shows the DNA sequence of the Polygalacturonase gene for Mutation 13342.
SEQ. ID. NO.: 50 shows the protein sequence encoded by SEQ. ID. NO.: 49.
The present invention describes: a series of independent non-transgenic mutations created in the polygalacturonase (PG) gene of tomato; tomato plants having these mutations in their PG gene; and a method of creating and identifying similar and/or additional mutations in the PG gene of tomato plants. The present invention further describes tomato plants exhibiting reduced PG enzyme activity and slower fruit softening post harvest without the inclusion of foreign nucleic acids in the tomato plants' genomes.
As shown in
In order to create and identify the PG gene mutations and slower softening tomatoes of the present invention, a method known as TILLING was utilized. See McCallum, et al., Nature Biotechnology (April 2000), 18: 455-457; McCallum, et al., (June 2000) Plant Physiology, Vol. 123, pp. 439-442; and U.S. Pat. No. 5,994,075, all of which are incorporated herein by reference. In the basic TILLING methodology, plant material, such as seeds, are subjected to chemical mutagenesis, which creates a series of mutations within the genomes of the seeds' cells. The mutagenized seeds are grown into adult M1 plants and self-pollinated. DNA samples from the resulting M2 plants are pooled and are then screened for mutations in a gene of interest. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the gene of interest.
Any cultivar of tomato having at least one PG gene with substantial homology to Seq. I.D. No. 1 may be used in the present invention. The homology between the PG gene and Seq. I.D. No. 1 may be as low as 60% provided that the homology in the conserved regions of the gene are higher. Thus one of skill in the art may prefer a tomato cultivar having commercial popularity or one having specific desired characteristics in which to create their PG-mutated tomato plants. Alternatively, one of skill in the art may prefer a tomato cultivar having few polymorphisms, such as an in-bred cultivar, in order to facilitate screening for mutations within the PG gene.
In one embodiment of the present invention, seeds from the tomato plant are mutagenized and then grown into M1 plants. The M1 plants are then allowed to self-pollinate and seeds from the M1 plant are grown into M2 plants, which are then screened for mutations in their PG genes. However, one of skill in the art would understand that a variety of tomato plant materials, including but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenized in order to create the PG-mutated tomato plants of the present invention. However, the type of plant material mutagenized may affect when the plant DNA is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant, the seeds resulting from that pollination are grown into M1 plants. Every cell of the M1 plants will contain mutations created in the pollen, thus these M1 plants may then be screened for PG gene mutations instead of waiting until the M2 generation.
Mutagens creating primarily point mutations and short deletions, insertions, transversions, and or transitions (about 1 to about 5 nucleotides), such as chemical mutagens or radiation, may be used to create the mutations of the present invention. For example, but not limited to, mutagens such as ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), triethylmelamine (1′EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N′-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9 [3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170), formaldehyde, and the like may be used to mutagenize the plant tissue in order to create the PG gene mutations of the present invention. Spontaneous mutations in the fruit PG gene that may not have been directly caused by the mutagen can also be identified using the present invention.
Any method of plant DNA preparation known to those of skill in the art may be used to prepare the tomato plant DNA for PG mutation screening. For example, See D. H. Chen and Ronald, P. C., Plant Molecular Biology Reporter 17: 53-57 (1999); C. N. Stewart and Via, L E, Bio Techniques, 1993, Vol. 14(5): 748-749. Additionally, several commercial kits are available, including kits from Qiagen (Valencia, Calif.) and Qbiogene (Carlsbad, Calif.).
The prepared DNA from individual tomato plants are then pooled in order to expedite screening for mutations in the PG genes of the entire population of plants originating from the mutagenized plant tissue. The size of the pooled group is dependent upon the sensitivity of the screening method used. Preferably, groups of four or more individuals are pooled.
After the DNA samples are pooled, the pools are subjected to PG gene-specific amplification techniques, such as Polymerase Chain Reaction (PCR). For a general overview of PCR, see PCR Protocols: A Guide to Methods and Applications (Inns, M., Gelfand, D., Sninsky, J., and White, T., eds.), Academic Press, San Diego (1990). Any primer specific to the PG gene or the sequences immediately adjacent to the PG gene may be utilized to amplify the PG genes within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the PG gene where useful mutations are most likely to arise. For example, the primer should maximize the amount of exonic sequence of the PG gene and, likewise, avoid intronic sequences of the gene. Additionally, it is preferable for the primer to avoid known polymorphism sites in order to ease screening for point mutations. Furthermore, when specifically screening for mutations that will knock out the PG enzymatic activity, it is preferable to target the end of the PG gene or to target areas of the PG gene that are highly conserved. To facilitate detection of PCR products on a gel, the PCR primer may be labeled using any conventional labeling method. Exemplary primers (SEQ. ID. Nos. 3-46) that have proven useful in identifying useful mutations within the PG gene sequence are shown below in Table 1.
The PCR amplification products may be screened for PG mutations using any method that identifies heteroduplexes between wild type and mutant genes. For example, but not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE) (Q. Li, et al, Electrophoresis, 23(10): 1499-1511 (May 2002), or by fragmentation using chemical cleavage, such as used in the high throughput method described by Colbert et al., Plant Physiology, 126:480-484 (June 2001). Preferably the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program.
Mutations that reduce PG enzyme activity in the plant are desirable. Preferred mutations include those that prematurely truncate the translation of the PG protein, such as those mutations that create a stop codon within the amino acid sequence of the PG protein. Additional preferred mutations include those that cause the mRNA to be alternatively spliced, such as mutations in and around the intron splice sites within the mRNA. Furthermore, any mutations that create an amino acid change within one of the fifteen highly conserved residues of the PG polypeptide are also preferred.
Once an M2 plant having a mutated PG gene is identified, then the mutations are analyzed to determine its potential affect on the expression, translation, and/or activity of the PG enzyme. First, the PCR fragment containing the mutation is sequenced, using standard sequencing techniques, in order to determine the exact location of the mutation in relation to the overall PG gene sequence. Second, in order to determine the severity of the change, a LOGO analysis is performed on the amino acid sequence BLOCK in which a mutation is located. Protein BLOCKS are multiply-aligned, ungapped segments corresponding to the most highly conserved regions of the protein families. Henikoff et al., Gene 163: GC17-GC26 (1995). LOGOs are a graphical representation of aligned sequences where the size of each amino acid residue is proportional to its frequency in that position. The LOGO for a BLOCK is calculated from the position-specific scoring matrix (PSSM). Tomato PG belongs to the glycoside hydrolase protein family 28 (BLOCK 1PB000743). One hundred and forty-seven members of this family were used to identify the seven conserved blocks within the family that are included in the BLOCKS database.
If the initial assessment of the mutation in the M2 plant appears to be in a useful position within the PG gene, then further phenotypic analysis of the tomato plant containing that mutation is pursued. First, the M2 plant is backcrossed twice in order to eliminate background mutations. Then the M2 plant is self-pollinated in order to create a plant that is homozygous for the PG mutation.
Physical and biochemical characteristics of these homozygous PG mutant plants are then assessed. Mutant PG tomatoes are evaluated for delayed softening compared to the normal (wild type) parental tomato lines. Normal fruit ripens such that the color of the tomato changes from light green to red. As this change happens, the fruit tends to become softer such that compression under a specified weight becomes greater and/or the force required to depress the surface of the fruit a specified distance becomes greater. See Cantwell, M. Report to the California Tomato Commission: Tomato Variety Trials: Postharvest Evaluations for 2001; Edan, Y., H. Pasternak, I. Shmulevich, D. Rachmani, D. Guedalia, S. Grinberg and E. Fallik. 1997. Color and firmness classification of fresh market tomatoes. J. Food Science 62(4): 793-796; Errington, N., J. R. Mitchell and G. A. Tucker. 1997. Changes in the force relaxation and compression responses of tomatoes during ripening: the effect of continual testing and polygalacturonase activity. Postharvest Biol. Tech. 11: 141-147; Lesage, P. and M-F. Destain. 1996. Measurement of tomato firmness by using a non-destructive mechanical sensor. Postharvest Biol. Tech. 8: 45-55.
The following mutations are exemplary of the tomato mutations created and identified according to the present invention. One exemplary mutation, correlates with a change of G to A at nucleotide 1969 of SEQ. ID. NO. 1, counting A in the ATG of the START CODON as nucleotide position 1. This mutation results in a change from glycine to arginine at amino acid 178 in the expressed protein. The change from glycine to arginine at 178 is a dramatic amino acid change both in terms of charge and size. The G 178R mutation is within block 13 of this family. As shown in
Another exemplary mutation, created and identified according to the present invention, correlates with a T to A change at nucleotide position 2940 of SEQ. ID. N0.1, counting A in the ATG of the START CODON as nucleotide position 1. This mutation results in a change from histidine to glutamine at amino acid 252. The H252Q mutation is within block D of the glycoside hydrolase protein family. As shown in
The following Examples are offered by way of illustration, not limitation.
In one embodiment of the present invention tomato seeds of cultivars 20 Shady Lady (hybrid) and NC 84173 (inbred line provided by R. Gardner at the University of North Carolina) were vacuum infiltrated in H2O (ca. 4 min. with ca. 1000 seeds/100 ml H2O). The seeds were then placed on a shaker (45 rpm) in a fume hood at ambient temperature. The mutagen ethyl methanesulfonate (EMS) was added to the imbibing seeds for final concentrations ranging from about 0.1% to about 1.6% (v/v). EMS concentrations of about 0.4 to about 1.2% were determined to be optimal for these studies. Following a 24-hour incubation, the EMS solution was replaced with fresh H2O (4ū to an est. EMS dilution 112,000,000,000). The seeds were then rinsed under running water for ca. 1 hour. Finally, the mutagenized seeds were planted (96/tray) in potting soil and allowed to germinate in the greenhouse. Four to six week old surviving plants were transferred to the field to grow to fully mature M1 plants. The mature M1 plants were allowed to self-pollinate and then seeds from the M1 plant were collected and planted to produce M2 plants.
DNA from these M2 plants was extracted and prepared in order to identify which M2 plants carried a mutation in their PG gene. The M2 plant DNA was prepared using the methods and reagents contained in the Qiagen« (Valencia, Calif.) 96 Plant Kit. Approximately 0.1 g of frozen plant sample was placed in a sample tube with a tungsten bead, frozen in liquid nitrogen and ground 2 times for 1 minute each at 20 Hz using the Qiagen« Mixer Mill MM 300. Next 400 μl solution API [buffer API, solution DX and RNAse (100 ug/ml)] at 80░ C. was added to the sample. The tube was sealed and shaken for 15 seconds. Following the addition of 130 μl buffer AP2, the tube was shaken for 15 seconds. The samples were then frozen for at least 10 minutes at minus 20░ C. The samples were then centrifuged for 20 minutes at 5600ūg. A 400 μl aliquot of supernatant was transferred to another sample tube. Following the addition of 600 μl of buffer AP3/E, this sample tube was capped and shaken for 15 seconds. A filter plate was placed on a square well block and 1 ml of the sample solution was applied to each well and the plate was sealed. The plate and block were centrifuged for 4 minutes at 5600ūg. Next 800 of buffer AW was added to each well of the filter plate, sealed and spun for 15 minutes at 5600ūg in the square well block. The filter plate was then placed on a new set of sample tubes and 100 μl of buffer AE was applied to the filter. It was capped and incubated at room temperature for 1 minute and then spun for 2 minutes at 5600ūg. This step was repeated with an additional 100 μl buffer AE. The filter plate was removed and the filtrates were pooled and the tubes capped. Then the individual samples were normalized to a concentration of 25 ng/μl.
The M2 DNA was pooled into groups of four or more individual plants each. For pools containing four individuals, the DNA concentration for each individual within the pool was 0.25 ng/μl with a final concentration of 1 ng/μl for the entire pool. The pooled DNA samples were arrayed on microtiter plates and subjected to gene-specific PCR.
PCR amplification was performed in 15 μl volumes containing 5 ng pooled or individual DNA, 0.75ū ExTaq buffer (Panvera, Madison, Wis.), 2.6 mM MgCl2, 0.3 mM dNTPs, 0.3 μM primers, 0.05 U Ex-Taq (Panvera, Madison, Wis.) DNA polymerase. PCR amplifications were performed using an MJ Research thermal cycler as follows: 95░ C. for 2 minutes; 8 cycles of ōtouchdown PCRö (94░ C. for 20 second, followed by annealing step starting at 70-68░ C. for 30 seconds decreasing 1░ C. per cycle, then a temperature ramp of 0.5░ C. per second to 72░ C. followed by 72░ C. for 1 minute); 25-45 cycles of 94░ C. for 20 seconds, 63-61░ C. for 30 seconds, ramp 0.5░ C./sec to 72░ C., 72░ C. for 1 minute; 72░ C. for 8 minutes; 98░ C. for 8 minutes; 80░ C. for 20 seconds; 60 cycles of 80░ C. for 7 secondsŚ0.3 degrees/cycle.
The PCR, primers (MWG Biotech, Inc., High Point, N.C.) were mixed as follows:
9 μl 100 μM IRD-700 labeled Left primer.
1 μl 100 μM Left primer.
10 μl 100 μM Right primer.
The IRD-700 label can be attached to either the right or left primer. Preferably, the labeled to unlabeled primer ratio is 9:1. Alternatively, Cy5.5 modified primers or IRD-800 modified primers could be used. The label was coupled to the oligonucleotide using conventional phosphoamidite chemistry.
For digestion of 15-μL PCR products in 96-well plates, 30 μL of a solution containing 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (pH 7.5), 10 mM MgSO4, 0.002% (w/v) Triton X-100, 20 ng mL−1 of bovine serum albumin, and 1/1000 dilution of CEL 1 (50 units μL−1) was added with mixing on ice, and the plate was incubated at 45░ C. for 15 min. CEL 1 was purified from 30 kg of celery as described by Oleykowski et al., Nucleic Acids Res 26: 4597-4602 (1998), except that Poros HQ rather than Mono Q was used, and the PhenylSepharose and Superdex 75 columns were omitted. The specific activity was 1ū106 units mL−1, where a unit is defined as the amount of CEL 1 required to digest 50% of 200 ng of a 500-bp DNA fragment that has a single mismatch in 50% of the duplexes. Reactions were stopped by addition of 5 μL 0.15 M EDTA (pH 8) and the mixture pipetted into wells of a spin plate (G50, Sephadex) prepared and spun according to the manufacturer's recommendations into a plate containing 1 to 1.5 μL of formamide load solution [1 mM EDTA (pH 8) and 200 μg mL1 bromophenol blue in deionized formamide]. The volume was reduced to a minimum by incubation at 80░ C. uncovered (30-40 min) and stored on ice, then transferred to a membrane comb using a comb-loading robot (MWG Biotech). Alternatively, the DNA samples could have been concentrated using isopropanol precipitation. The comb was inserted into a slab acrylamide gel, electrophoresed for 10 min, and removed. Electrophoresis was continued for 4 h at 1,500-V, 40-W, and 40-mA limits at 50░ C.
After electrophoresis, the gel was imaged using a LI-COR (Lincoln, Nebr.) scanner which was set at a channel capable of detecting the IR Dye 700 label. The gel image showed sequence-specific pattern of background bands common to all 96 lanes. Rare events, such as mutations, created a new band that stood out above the background pattern. Plants with bands indicative of mutations of interest were evaluated by sequencing individual PCR products. Plants carrying mutations confirmed by sequencing were grown up as described above (e.g., the M2 plant was backcrossed twice in order to eliminate background mutations and self-pollinated in order to create a plant that was homozygous for the mutation).
Tomatoes Selected for Study:
Individual tomatoes selected for study were picked from plants derived from siblings of the same cross to preserve background phenotypes as much as possible. The plants and fruit were genotyped as homozygous for the mutation, heterozygous for the mutation, or wild type. Genotyping was performed using a genetic method for determining single base pair mismatches referred to in the scientific literature as ōdCAPingö, see M. M Neff et al., The Plant Journal 14:387-392 (1998). Briefly, a degenerate PCR oligonucleotide is designed to create a restriction endonuclease recognition site when the mutant base pair is present. Plants are then simply genotyped using a PCR reaction followed by a restriction enzyme digestion and then analysis on an agarose gel.
A test was devised to simulate consumer perception of tomato fruit firmness in the three genotypes of the 13345 mutant. Fruits were evaluated at the red ripe stage. Four fruits of each genotype were blindly labeled, and 15 people were asked to rank each set as most firm, least firm, or in between (mid firm). Of the people surveyed, 80% ranked the homozygous PG mutant tomatoes as the most firm; 20% ranked the heterozygous mutant tomatoes as the most firm; and no one ranked the wild type as the most firm. Results are shown in
Objective color values were determined for table-ripe wild type and mutant 13345 and 13342 tomatoes using a Minolta Color meter. Data were reported as ōhueö and from 20-30 hue values were measured. Hue is the single most useful color value and the lower the hue value, the redder the tomato. In support of the idea that some characteristics associated with ripening do not differ, the results showed that PG mutants (hue values of 35.8 and 34.5) were similar in color to wild type tomatoes (hue value of 36.6).
Polygalacturonase enzymatic activity was measured spectrophotometrically using two different in vitro color assays that quantify the formation of reduced sugars from a polygalacturonic substrate. One assay utilized 3,5-dintrosalicylate (DNS) for color detection, and was performed as in Redenbaugh K, Hiatt W, Martineau B, Kramer M, Sheehy R, Sanders R, Houck C, and Emlay D. Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the Flavr Savr Tomato. CRC Press (1992); R. Sheehy, et al., PNAS 85:8805-8809 (1988); Z. M. Ali and C. J. Brady, Aust. J. Plant Physiol. 9:155-169 (1982). The other assay utilized bicinchoninic acid (BCA) as the color substrate and was performed as in G. E. Anthon et al., Journal of Agricultural and Food Chemistry 50:6153-6159 (2002); and D. Fachin, et al., Journal of Food Science 67:1610-1615 (2002).
Briefly, cell wall extracts from individual tomatoes were prepared as follows: tomatoes were sliced, locular tissue and seeds were removed, and 100 grams of the remaining tomato tissue were homogenized in 300 milliliters (ml) cold H2O and centrifuged at 4000 rpm in a tabletop centrifuge. The pellet was resuspended in 300 ml extraction buffer (1.7 M NaCl, 40 mM β-mercaptoethanol, 50 mM sodium phosphate, pH 4.6) and stirred for 4 hours at 4░ C. The suspension was then centrifuged as before and the supernatant was reserved for use in the DNS color assay. Because PG enzyme is the predominant protein in the cell wall extracts, any variation in PG protein amount due to genotype would interfere with using protein concentration in the normalization process, thus in the 13345 mutant where PG protein is absent the lysates were instead normalized to wet weight of starting material.
For the color assay, 0.1 ml 2M ammonium chloride, 1 ml 1% polygalacturonic acid, and 0.1 ml cell wall extract were mixed together in tubes on ice. Samples were vortexed and a small amount was reserved as a control for the amount of reduced sugars present prior to incubation with PG enzyme. The remainder of each sample was incubated at 37░ C. for 2 hours. After incubation, samples were place on ice and 0.1 ml of each was transferred to a new tube at room temperature with 0.2 ml DNS color reagent (1 g DNS/20 mls 2M NaOH, 30 g sodium potassium tartarate/50 mls warm water; the two reagents are then combined and diluted to 100 mls with warm water). Tubes were boiled in a water bath for 5 minutes and then 2 mls H2O added to each tube. Tubes were spun to clarify and then read at an absorbance of A540 on a spectrophotometer.
Results of the DNS based PG activity assay, shown in
Briefly, cell wall extracts from individual tomatoes were prepared as follows: tomatoes were sliced, locular tissue and seeds were removed, and 15 g of the remaining tissue was homogenized in 30 ml cold H2O. ś7.5 mls 1N HCl was added (to a final pH of 3.0), and the homogenate was spun at 4000 rpm in a tabletop centrifuge. The pellet was washed in 30 mls cold H2O, and spun as before, The washed pellet was then resuspended in 7.5 mls extraction buffer (0.1M sodium phosphate pH 6.5, 1.2M NaCl) and incubated on ice for 30 minutes. The suspension was spun as before, and the supernatant was reserved for use in the BCA color assay. Again, the lysates were normalized to wet weight starting material in the 13345 mutant, and protein concentration in the 13342 mutant.
For the BCA color assay, 0.5 ml 1% polygalacturonic acid, 0.2 ml 1M NaCl, 1.3 ml H2O, and 10 μL extract were mixed together and incubated at 37░ C. for 30 minutes. After incubation, 1 ml carbonate buffer (54.3 g/L disodium carbonate/24.2 g/L sodium monocarbonate) was added to terminate each reaction. 0.6 mls each terminated reaction was then added to 1.9 ml H2O and 1.6 mls color reagent (equal volumes reagents A and B where reagent A is 151.96 g bicinchoninic acid/L H2O and reagent B is 1.24 g/L CuSO4ŚH2O, 1.26 g/L L-serine), and incubated at 80░ C. for 30 minutes. Color development was then measured at A560 using a spectrophotometer.
Results of the BCA based PG activity assay, shown in
To ascertain the amounts of PG enzyme in the mutant tomatoes relative to wild type tomatoes, cell wall extraction lysates from the activity assays were run on SDS-PAGE gels and visualized both by Coomassie stain and by Western blot using a PG-specific polyclonal antibody as in D. DellaPenna et al., PNAS, 83:6420-6424 (1986). (PG antibody was a generous gift of Dr Alan Bennett, University of California, Davis).
Results of the Western blot, shown in
DNA from tomato plant 13345, originating from seeds of cultivar Shady Lady that were incubated in 1.2% EMS, was amplified using primer pair PGL3 (SEQ. ID. NOs. 023 and 043). The PCR amplification products were then incubated with CEL 1 and electrophoresed. The electrophoresis gel image showed a fragment at the approximate position of 204 bp, above the background pattern for the PCR amplification products. Therefore, it was likely that this fragment contained a heteroduplex created by a mutation in the PG gene. Sequence analysis of this fragment showed that the mutation was associated with a G to A change at nucleotide 1969 of SEQ. I.D. No. 1, counting A in the ATG of the START CODON as nucleotide position 1. This mutation correlates with a change from glycine to arginine at amino acid 178 of the PG polypeptide.
This mutation is within block B of the glycoside hydrolase protein family. LOGO analysis of the G178R mutation within this block revealed that the mutation lies at one of the fifteen most conserved amino acids within the family.
Tomato fruits containing Mutation 13345 exhibited lower PG enzyme activity compared to their wild type sibling, and were considered firmer than the wild type sibling.
Tomato plant 13342, originating from seeds of cultivar Shady Lady that were incubated in 0.6% EMS, was screened with primer pair PGL9 (SEQ. ID. NOs. 027 and 039). The PCR amplification products were then incubated with CEL 1 and electrophoresed. The electrophoresis gel image showed a fragment at the approximate position of 385 bp, above the background pattern for the PCR amplification products. Therefore, it was likely that this fragment contained a heteroduplex created by a mutation in the PG gene. Sequence analysis of this fragment showed the mutation was associated with a T to A change at nucleotide 2940 of SEQ. I.D. No. 1, counting A in the ATG of the START CODON as nucleotide position 1. This mutation correlates with a change from histidine to glutamine at amino acid 252 of the PG polypeptide.
Tomato fruits containing Mutation 13342 exhibited lower PG enzyme activity compared to their wild type sibling, and were considered firmer than their wild type sibling.
The above examples are provided to illustrate the invention but not limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims and all their equivalents. All publications, patents, and patent applications cited herein are hereby incorporated by reference.
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Morning Show Follower Shares Picture Illustrating Canadian Cold
Updated: 01/03/2014 - One of the Morning Show's Twitter followers shared a picture showing just how cold it is in parts of Canada this week!
Updated: 01/03/2014 - One of the Morning Show's Twitter followers shared a picture showing just how cold it is in parts of Canada this week!
Updated: 01/02/2014 - One of the Morning Show's Twitter followers shared a picture of a rainbow from Wales!
Updated: 01/02/2014 - It was a very cold last day of 2013 in the city of Winnipeg, Canada. It was minus 31 degrees Tuesday.
Updated: 01/01/2014 - Tuesday, December 31st, 2013 marked the end of the preliminary rounds of the 2014 IIHF World Junior Hockey Championships.
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Updated: 12/31/2013 - The category three system is expected to bring massive storm surges of up to five metres, flooding and destructive winds of about 200 kilometres an hour.
Posted: 12/31/2013 - The 2014 IIHF World Junior Hockey Championships continued on Monday in Sweden.
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Posted: 12/30/2013 - The preliminary rounds of the 2014 IIHF World Junior Hockey Championships continued over the weekend in Sweden.
Posted: 12/29/2013 - Capitol Dome Renovations... A week after ice storm hits Maine, more than 4,000 still without power... Federal government ends extended jobless benefits... Health law to put calorie info on vending machines... Boy Scouts open ranks to gay youth on Jan. 1... Despite files, Conn. school shooter remains enigma... Kerry to return next week to Middle East... Police: New Mexico mom admits to kicking child... Pregnant nurse fired in Pa. over flu shot... Pa. man admits beating `Amish Mafia' star... Death of man in Border Patrol custody investigated... Separate avalanches in Wyoming kill 2 within hours... Times Square preps for New Year's Eve bash...
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Updated: 12/29/2013 - On Dec 28 - In 1170, Thomas Becket, the Archbishop of Canterbury, was slain in Canterbury Cathedral by knights loyal to King Henry II... In 1890, the Wounded Knee massacre took place in South Dakota as an estimated 300 Sioux Indians were killed by U.S. troops sent to disarm them... In 1916, Grigory Rasputin, the so-called "Mad Monk" who'd wielded great influence with Czar Nicholas II, was killed by a group of Russian noblemen in St. Petersburg... In 1940, during World War II, Germany dropped incendiary bombs on London, setting off what came to be known as "The Second Great Fire of London."... In 1972, Eastern Air Lines Flight 401, a Lockheed L-1011 Tristar, crashed into the Florida Everglades near Miami International Airport, killing 101 of the 176 people aboard...
Updated: 12/27/2013 - Many parents despair of what to do with a house full of unwanted toys, especially post Christmas when another load has just been unwrapped.
Updated: 12/27/2013 - The 2014 IIHF World Junior Hockey Championships got underway on Thursday in Sweden.
Updated: 12/26/2013 - Heavy snow continues to fall in parts of Canada, and new pictures from New Brunswick in that country show just how deep the snow has accumulated.
Updated: 12/26/2013 - City workers and university students alike discharge the pressures of daily life in a night of live music and a whirlwind of pillows on Christmas day (December 25).
Updated: 12/26/2013 - It's a wrap for NASA's Christmas Eve spacewalk.
Posted: 12/22/2013 - Memorials to mark Lockerbie attack 25 years later... UN helicopter hit, downed in S. Sudan... Attacks across Iraq kill 15 people, including senior military commander... Syrian rebels seize strategic hospital in Aleppo... China lashes out at Japan's new defense plan... Saudi Arabia reports 1 more death from new virus... Gunmen kill anti-polio worker in NW Pakistan... UN urges more aid for Philippine typhoon recovery... Raul Castro issues stern warning to entrepreneurs... Arab League rejects US proposal in Mideast deal... 'Topless' protest falls flat in Rio de Janeiro...
Updated: 12/22/2013 - Today's Highlight in History: On Dec. 22, 1944, during the World War II Battle of the Bulge, U.S. Brig. Gen. Anthony C. McAuliffe rejected a German demand for surrender, writing "Nuts!" in his official reply.
Posted: 12/20/2013 - Plastic banknotes are to be brought into circulation in 2016, the Bank of England chief announced on Wednesday (December 18).
Updated: 12/19/2013 - The European Space Agency is preparing to launch a satellite that will produce the most accurate three-dimensional map of our part of the Milky Way.
Updated: 12/19/2013 - It's the busiest time of the year for Santa and his elves. The bearded man is supervising the wrapping of all of the presents.
Updated: 12/18/2013 - A shopping mall in Bangkok has claimed this year's crown as the world's most photographed location on Instagram.


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Leahy joins brief on DOMAMarch 04,2013MONTPELIER Vermont U.S. Sen. Patrick Leahy is joining more than 200 other members of Congress in urging the U.S. Supreme Court to strike down the federal Defense of Marriage Act.
The court is soon to hear the case of Edith Windsor, who had to pay $373,000 in taxes on her spouse Thea Spyers estate after Spyer died because the federal government didnt recognize that the New York women were married.
A brief filed Friday includes testimony from Vermont marriage equality advocate Susan Murray, who argued that DOMA works to erode the financial security of same-sex couples.MORE IN Vermont NewsHYDE PARK A lawyer for a sheriffs deputy who saw a domestic abuse investigation against him... Full StoryMONTPELIER The Vermont Board of Education is seeking public comment as members draft a... Full StoryPolice: Snowball Full Story
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See you political class again trying to narrow the concerns of immigration to benefit claiming. I can assure you this don't wash our concerns yes...
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Game|Life3 seasons, 26 episodes
Video game experts Chris Kohler, Peter Rubin, and Chris Baker dissect the world of games with the expertise of professionals and the excitement of die-hard fans. Famed designers and players join the team to talk about the latest news and what's great—and not-so-great—in the industry.
Special Edition: Atari E.T. Excavation
Gaming The Making of the "Atari: Game Over" Documentary with Zak Penn It's been called the worst video game in history, but could E.T. the Extra-Terrestrial really be the cause of Atari's downfall? In the new documentary "Atari: Game Over" director Zak Penn follows the rise and fall of the trailblazing video game company, including the game that caused Atari's demise. Watch as Penn takes us behind the scenes of the documentary and shows us exclusive footage ahead of the November 20 release.
Gaming Teaser: Excavating the Atari E.T. Video Game Burial Site In 1983 after the critical and commercial failure of Atari's E.T. the Extra-Terrestrial video game, the company allegedly dumped millions of cartridges into a landfill in Alamogordo, N.M. Now, more than 30 years later, the site is being excavated and filmed for an upcoming docu-series by The Incredible Hulk screenwriter Zak Penn. The day before the big dig we headed south to talk with Penn about the urban legend. Plus, check out our exclusive first look at the excavation site.
Gaming Excavating the Atari E.T. Video Game Burial Site It's been dubbed The Great Video Game Burial of 1983, but did Atari really toss millions of cartridges of its biggest flop: E.T. the Extra-Terrestrial? Chris Kohler is onsite to witness an excavation of the landfill to see if the joke's really on us, and to talk with those involved with the project, including original game designer Howard Scott Warshaw.


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i heard a brewerey served choc.coved starwberry beer. A mix between the choc. milk stoudt and strawberry wheat. Just put both in the keg and waiting to carb up. Just wondering if anyone knows the best way to pour it. i.e like a black n tan, or layer, just mix? thanks
I read that knew you must be talking about LBC. I haven't tried their mix yet, but maybe the next time I stop in. I think you would just pour them both in and let them mix. The black and tan layering would only work if your milk stout was on nitrogen.
Draft 1: Lightly smoked Weizen
Draft 2: Belgian Wit
Draft 3: Rauchbier
Primary 1: German Scwarzbier
Primary 2:
On Deck: Belgian Brown Ale, Pale Ale, Stout
im about 30 min from there i haven't tried it yet but hear great things about it


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Apricot, sesame and cranberry biscuits
Dried apricots and cranberries add wholesome sweetness to these delicious biscuit treats.
To Prep
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recipe To my
- Ingredients
- Nutrition
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All nutrition values are per serve.
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I left out the apricots, increased cranberries to 1 cup, and used spelt flour. I had to add 3 heaped soup spoons of extra flour to make these stiff enough, forgot that spelt flour takes less moisture up! The biscuits were still very soft after the recommended cooking time, I gave nearly the same time again but turned the oven down to about 160C. That worked well. Will make these again.
I made a couple of changes, I substituted spelt for regular flour and added a handful of white chocolate chips, very yummy.
Used sultanas instead of dried apricots, added a couple of tablespoons of coconut to mixture. Found it easy to roll into balls, didn't have any problems with wet hands. Didn't find them too sweet, they were quite nice. Would make these again for sure.
YUM! And so quick and easy! Didn't have quite enough sesame seeds or dried apricots so topped up each quantity with some coconut and sultanas. Lovely eaten while still warm.
I replaced the butter with 75ml light olive oil and the mixture made 25 biscuits, however it was very sticky - roll it with wet hands.
easy to make and taste great! swapped the apricot for white choc chips!!
Delicious! I substituted golden syrup for honey. Easy and quick!
Quite yummy, quick & easy. I had no sesame seeds, so substituted finely chopped macadamia nuts, worked well.
melt n mix.. the best! i substituted choc bits (less healthy, but yummier!) for the apricot, and dessicated coconut for the sesame seeds. delicious. :)
Yummo! Have made these bikkies a couple of times now and everyone absolutely loves them. If you like soft Anzacs - these are for you! Make sure your hands are wet when rolling out the biscuits, otherwise things get very sticky.
made ths recipe tonight as we ran out of bikkies now for smokos..made 2 lots of mixture but the sesame seeds weren't enough...anyway, i'm so sorry but we didn't like the taste, maybe there's still an ingredient missing & i reckon this can be a good bikie for those who are trying to lose weight...found it too fiddly/sooo sticky to make. didn't have craisins just used up sultanas(as that's what i only got in my cupboard)... :-(
Easy to make but way too sweet for my liking. A bit too moist. It doesn't have the "biscuit" texture that I was hoping for. However, I am a horrible cook so it is probably more of the cooks fault than the receipes!
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Transformers Sequel Bashes Box Office
Ticket sales were good but far from spectacular for Tuesday's late-night screenings of Transformers Dark of the Moon , studio figures indicated. The movie earned $13.5 million, but only $5.5 million -- 41 percent -- from 3D screens, suggesting that, given premium pricing for 3D tickets, fewer than a third of moviegoers elected to see it in that format. (A conflicting report said that the $5.5 million came from 3D showings at 9 00 p.m. and $8.8 million from both 3D and 2D screenings at midnight.) Moreover, the result falls short of the midnight screenings for the last Transformers movie, Revenge of the Fallen in 2009, which took in $16 million without 3D premium pricing going for it. Paramount now says that it expects the movie to earn $160 million over the six-day holiday, which many industry analysts regard as a modest forecast, particularly given the fact that Fallen earned $215 million in its first six days. UPDATE Daily Variety reported this morning (Thursday) that Transformers Dark of the Moon earned $37.32 million on Wednesday, including $8 million from midnight screenings. That put it ahead of Pirates of the Caribbean On Stranger Tides 's $35 million to make it the biggest opening-day gross for any film this year. (That would put it far behind Fallen 's $62.02 million debut.) The trade paper also reported that the film made $79.4 million overseas, including previews.


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The third Monday in January is popularly known in Hollywood circles as "hungover-as-shit Monday," as it immediately follows the Golden Globes, the award season's drunker, more badly-behaved little cousin. Among all the questions raised by the Golden Globes last night (What was the point of asking a ...Read More »
We could stretch it to more than four, but let's stay focused. All ears, of course, will be on Ricky Gervais, as we wonder what he can possibly do to top his hilarious, irreverent turn as host last year. The first five minutes should tell.Read More »
It's kind of cool to hear about the creation of this scene. As Meryl Streep says: " Margaret Thatcher really did break ground because she showed a woman could be a leader." The film opens in the UK today and will roll out further here in the US next week.Read More »
Some good news for Meryl Streep and The Iron Lady. First, the box office for the film which opened December 30 was terrific. It grossed $280,409 in four theatres (in NY and LA) for an amazing total of $70,102 per screen. Not surprising, the film skewed female and older. 54% of the attendees were...Read More »
You would think that with 16 Academy Award nominations (2 wins), 26 Golden Globe nominations (7 wins), 2 Emmy Awards, 2 Screen Actors Guild Awards, a Cannes Film Festival award, 5 New York Film Critics Circle Awards, 5 Grammy Award nominations, a BAFTA award, an Australian Film Institute Award, a To...Read More »
The Berlinale's honorary lifetime achievement award, the Golden Bear, will be given to "The Iron Lady"'s Meryl Streep on February 14. The Berlin fest's Dieter Kosslick calls Streep "a brilliant, versatile performer who moves with ease between dramatic and comedic roles."Read More »
The 62nd Berlin International Film Festival has announced that Meryl Streep will receive an Honorary Golden Bear February 14. The ceremony will take place in conjunction with a screening of her latest work, "The Iron Lady," at the Berlinale Palast.Read More »
The Iron LadyRead More »
It goes without saying that Meryl Streep is always worth watching; in the case of 'The Iron Lady', her uncanny performance as Margaret Thatcher is the best, and possibly only, reason to see this pallid biography. Screenwriter Abi Morgan does provide a primer on Thatcher's remarkable rise from grocer...Read More »
Well, these are not the reviews Harvey Weinstein had in mind when he backed "The Iron Lady." So far the movie is scoring 64% on Rotten Tomatoes. Stateside critics are praising Meryl Streep's impeccable portrait of a much-revered and reviled British leader, while many question Phyllida Lloyd's direct...Read More »