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Later this month, a number of nations, including the U.S., will descend upon Paris, France for a week and a half to discuss global efforts to reduce greenhouse gas emissions. The 2015 United Nations Climate Change Conference will take place from November 30th to December 11th, and while most of the event will be used to discuss new ways to mitigate the effects of global climate change, President Obama will be gearing up to discuss the new additions to his climate plan. However, while the commander-in-chief is hoping that a bold climate agenda will spur other nations to take on or continue their own ambitious climate initiatives, the potential economic repercussions may create more of a dissuasive tone; especially for developing industrial nations such as China.
Dear FreedomWorks member, As one of our million-plus FreedomWorks members nationwide, I urge you to contact your senators and urge them to vote oppose ratifying the United Nations Convention on the Rights of Persons with Disabilities (CRPD).While the Senate recently voted to not ratify the CRPD, the U.N. treaty is far from dead— Senate leadership has already pledged to bring it to another vote at the beginning of the 113th Congress. The U.N. treaty may sound well-intentioned but it would erode U.S. sovereignty and hurt families with disabled children.
Dear FreedomWorks member, As one of our million-plus FreedomWorks members nationwide, I urge you to contact your senator and ask him or her to sign a letter by Sen. Jim DeMint (R-SC) in opposition to the United Nation's Law of the Sea Treaty (LOST). Congress is once again debating signing this international treaty that establishes a comprehensive set of rules governing the seas. All senators should take a principled stance against this unconstitutional treaty that threatens our national sovereignty and conflicts with basic free market principles.
Contact your congressman and tell them to stop flying first class on our dime.
IRS Commissioner John Koskinen has ushered in an era of deception and lawlessness.
Tell Republicans to end the special ObamaCare exemptions for Congress!
Mitch McConnell has lied to you and sided with the Democrats and lobbyists on the Ex-Im Bank. Take action!
Bureaucrats at the Export-Import Bank have been picking winners and losers in the marketplace for over 80 years. It's time to keep it shutdown!
Let's repeal ObamaCare. Let's force Obama to look Americans in the eye and veto a repeal that the majority of Americans want.
The REINS Act would cut down on Obama's regulations and return lawmaking power back to Congress.
Tell your representatives in Congress to stand against a federal gas tax increase.


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I mistakenly picked up a can of Campbell's Cheddar Cheese soup when I meant to get Cream of Chicken. But now I have the cheese soup in the cupboard, waiting to be used. Does anyone have a good recipe for it? I searched the Campbell's website and nothing really caught my eye. Thanks!
What to do with Cheddar Cheese Soup?
My friend recently made beer cheese soup. Not sure what all was in it exactly but google could have it...
Even M I R A C L E S take a little time.
Even M I R A C L E S take a little time.
I've been making this since I was 8! It was the first thing I ever made by myself :)
Potato, ham, and cheese casserole:
Combine in a casserole dish:
2-3 large sliced potatoes
1 chopped white onion
1 small diced ham (I usually use turkey ham)
1 can cheddar cheese soup
1 cup milk
salt and pepper to taste
Bake covered for one hour at 375. Super easy and really yummy!
I just dump in a pan add half a can if milk and bring it to warm.. then serve with cauliflower and broccoli!
*Cat in the Hat* 11y, *Thing She 1* 8y, *Thing He 2* 8y
Here are a couple ideas:
Mexican Chicken
Amy - amylz
Recipe courtesy Paula Deen
Vegetable oil cooking spray
1 (10 3/4-ounce) can cream of chicken soup
1 (10 3/4-ounce) can cheddar cheese soup
1 (10 3/4-ounce) can cream of mushroom soup
1 (10-ounce) can tomatoes
1 whole chicken, cooked, boned, and chopped or 4 cups leftover cooked chicken
Unsalted butter, for greasing pan
1 (11 1/2-ounce) package flour tortillas
2 cups shredded Cheddar
Preheat the oven to 350 degrees F. Spray a 13 by-9-inch pan with the cooking spray.
In a large bowl, stir together the 3 kinds of soup and the tomatoes. Stir in the chicken.
Layer the tortillas and the chicken mixture in the pan, beginning and ending with tortillas. Sprinkle the cheese over the casserole and bake for 30 minutes.
4 boneless skinless chicken breasts (frozen)
1 (16 ounce) carton fresh mushrooms, sliced
1 (10 3/4 ounce) can cream of chicken soup
1 (10 3/4 ounce) can cheddar cheese soup
1 bunch fresh broccoli, cut into flowerets
1/2 tsp dried minced garlic
1/2 tsp dried minced onion
2 cups uncooked white rice
2 cups cheddar cheese
Layer the crockpot with the still-frozen chicken, the broccoli, and the mushrooms. Whisk together the two soups with the garlic and onion, and pour over all. Cover and set on high for 4-6 hours, or low for 6-8 hours. Prepare rice as directed and add to crockpot. Add cheese and let simmer another 30 minutes.
Mom to 2 boys (13&9)
pizza hot dish
1lbs hamburger
1 16 oz jar of spaghtti sauce
1 8oz can of tomatoe sauce
1 can of chedder cheese soup
1 tsp of garlic
1 tsp of basil
and salt and pepper mix
spiral noodles
mix sauce and meat together mix noodles i add mozzeala and chedder cheese and bake 350 or 30 min
i also make quick Cheeseburger bake
1lb of hamburger
2 cup bisquick
1/4 cup milk
3/4 cup water
1 bag of mixed veggies
1 can chedder cheese soup
shredded chedder or american cheese
fry hamburger mix in soup milk veggies, then mix together bisquick and water spread on bottom of 9x13 baking dish top with meat mixture and top with cheese bake at 400 or 30 mins
Mock Cheese Fondue (Apple Juice) (details here)
2 cans (10 3/4 ounces each) condensed Cheddar cheese soup
2 cloves garlic, minced
4 egg yolks, beaten
1/2 cup apple cider or juice
Dippers (below)
In earthenware fondue pot, heat soup and garlic over low heat to just below boiling; remove from heat. Stir half of hot soup into egg yolks*; blend into remaining soup. Stir in cider; heat through, stirring constantly. Transfer pot to source of heat at table. Adjust heat when necessary to keep fondue warm. Swirl dippers in fondue. 4 servings.
Dippers: Cut-up cooked frankfurters, cooked Mini Meatballs, dill pickle chunks, cut up French bread and cauliflowerets.
I've used the cheese soup mixed with salsa and chicken to make quisedilla's
Vixkin Mom to Bugga bugga (11) and bumpydump (9)
Mac and Cheese
Kim *~*JoCaPa*~*
(this is from the Mueller's box) but I make it with Barilla Plus. I add 1 can of Campbell's Cheddar cheese soup too. This is a huge hit!!!!
2 tbsp (30 ml) cornstarch
1 tsp (5 ml) salt
1/2 tsp (2 ml) dry mustard (optional)
1/4 tsp (1 ml) pepper
2-1/2 cups (600 ml) milk
2 tbsp (30 ml) Margarine or butter
2 cups (475 ml) (8 oz (224 grm).) shredded American or Cheddar cheese, divided
8 oz (224 grm). Mueller's Elbows (about 2 cups), cooked 5 minutes and drained
* 1 can Campbell's Cheddar cheese soup
In medium saucepan combine corn starch, salt, dry mustard and pepper; stir in milk.
Add margarine.
Stirring constantly, bring to a boil over medium-high heat and boil 1 minute.
Remove from heat.
Stir in 1-3/4 cups (425 ml) cheese until melted.
Add * 1 can Campbell's Cheddar cheese soup
Add elbows.
Pour into greased 2-quart casserole.
Sprinkle with reserved cheese.
Bake uncovered in 375 degree (200 C.) oven 25 minutes or until lightly browned.
For the Mexican Variation-
Homemade Hamburger Helper Recipe
hmschlin2 aka Jen :)
1 onion, minced
1 lb any ground meat
1 can cream of whatever soup
1 1/3 cup water
1 ½-2 Cups smallish pasta or egg noodles
½ -1 Cup dairy product
Saute the onion and brown the ground meat, draining if necessary. Add cream of whatever soup and water, along with any flavoring. Bring to boil and add pasta. Cook on medium covered for 5 minutes and then uncovered until pasta is tender and desired consistency is achieved. Add dairy product and allow to melt/mingle for a few minutes. Serve.
Combination Ideas:
Stroganoff- Ground beef, cream of mushroom soup, egg noodles, ½ Cup sour cream, 1 tsp worchestshire for flavor
Creamy Cheesy Chicken-ground chicken, cream of chicken soup, tri color ruffles, 1 Cup cheddar cheese, 1 tsp dried rosemary for flavor
Mexican-ground beef or pork, cheddar cheese soup, egg noodles, 1 C cheddar jack cheese, 1 TBSP chili powder for flavor
BBQ beef-ground beef, tomato soup, macaroni, 1 C cheddar cheese, ¼ cup BBQ sauce for flavor
Tater Tot Casserole
Jaci (smallpotatoes)
1 1/2 lbs. ground beef
1 can cond. cheddar cheese soup
3/4 cup milk
1 1/4 cup sour cream
1 lb. Velveeta, cubed
1 cup shredded cheddar cheese.
1 28 oz. bag tater tots
Brown ground beef, drain. Add soup, milk, sour cream & Velveeta to skillet. Heat until melted. Remove from heat & stir in tater tots & pour into a greased 9x13 dish. Bake at 350 for 25 minutes. Add shredded cheese & bake for 5 more minutes or until cheese is melted.

subset: commoncrawl


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Voss named GPAC Player of the YearPublished by on
Former Worthington Trojan Jalen Voss has gathered up some serious hardware this season. The former Trojan boys basketball standout, now a senior at Dakota Wesleyan University in Mitchell (SD), was recently named the Great Plains Athletic Conference Player of the Year. In his senior season Voss averaged 21 points and 5.8 rebounds per game. He was named the GPAC Player of the Week 3 times during his senior season. Voss was named to the All-GPAC First Team this season and has been a First Team selection the last 3 seasons.


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|Efficacy of lamina terminalis fenestration in reducing shunt-dependent hydrocephalus following aneurysmal subarachnoid hemorrhage: a systematic review. Clinical article.
MedLine Citation:
|PMID: 19284236 Owner: NLM Status: MEDLINE
|OBJECT: Chronic hydrocephalus requiring shunt placement is a common complication following aneurysmal subarachnoid hemorrhage (SAH). Controversy exists over whether microsurgical fenestration of the lamina terminalis during aneurysm surgery affords a reduction in the development of shunt-dependent hydrocephalus. To resolve this debate, the authors performed a systematic review and quantitative analysis of the literature to determine the efficacy of lamina terminalis fenestration in reducing aneurysmal SAH-associated shunt-dependent hydrocephalus. METHODS: A MEDLINE (1950-2007) database search was performed using the following keywords, singly and in combination: "ventriculoperitoneal shunt," "hydrocephalus," "subarachnoid hemorrhage," "aneurysm," "fenestration," and "lamina terminalis." Additional studies were manually singled out by scrutinizing references from identified manuscripts, major neurosurgical journals and texts, and personal files. A recent study from the authors' institution was also incorporated into the review. Data from included studies were analyzed using the chi-square analysis and Student t-test. The Cochran-Mantel-Haenszel test was used to compare overall incidence of shunt-dependent hydrocephalus. RESULTS: The literature search revealed 19 studies, but only 11 were included in this review, involving 1973 patients. The fenestrated and nonfenestrated cohorts (combined from the various studies) differed significantly with regard to patient sex, age, and clinical grade as well as aneurysm location (p=0.0065, 0.0028, 0.0003, and 0.017, respectively). The overall incidence of shunt-dependent hydrocephalus in the fenestrated cohort was 10%, as compared with 14% in the nonfenestrated cohort (p=0.089). The relative risk of shunt-dependent hydrocephalus in the fenestrated cohort was 0.88 (95% CI 0.62-1.24). CONCLUSIONS: This systematic review revealed no significant association between lamina terminalis fenestration and a reduced incidence of shunt-dependent hydrocephalus. The interpretation of these results, however, is restricted by unmatched cohort differences as well as other inherent study limitations. Although the overall literature supports lamina terminalis fenestration, a number of authors have questioned the technique's benefits, thus rendering its efficacy in reducing shunt-dependent hydrocephalus unclear. A well-designed, multicenter, randomized controlled trial is needed to definitively address the efficacy of this microsurgical technique.
|Ricardo J Komotar; David K Hahn; Grace H Kim; Robert M Starke; Matthew C Garrett; Maxwell B Merkow; Marc L Otten; Robert R Sciacca; E Sander Connolly
Related Documents :
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19001456 - Impact of 3-mm blalock-taussig shunt in neonates and infants with a functionally single...
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Publication Detail:
|Type: Journal Article; Research Support, Non-U.S. Gov't; Review
Journal Detail:
|Title: Journal of neurosurgery Volume: 111 ISSN: 0022-3085 ISO Abbreviation: J. Neurosurg. Publication Date: 2009 Jul
Date Detail:
|Created Date: 2009-07-02 Completed Date: 2009-08-20 Revised Date: -
Medline Journal Info:
|Nlm Unique ID: 0253357 Medline TA: J Neurosurg Country: United States
Other Details:
|Languages: eng Pagination: 147-54 Citation Subset: AIM; IM
|Department of Neurological Surgery, Columbia University, New York, NY 10032, USA. firstname.lastname@example.com
Export Citation:
|APA/MLA Format Download EndNote Download BibTex
|MeSH Terms
Hydrocephalus* / etiology, prevention & control, surgery
Subarachnoid Hemorrhage / complications*, surgery*
Ventriculoperitoneal Shunt*
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
Previous Document: Intranasal delivery of erythropoietin plus insulin-like growth factor-I for acute neuroprotection in...
Next Document: Relevance of intracranial hypertension for cerebral metabolism in aneurysmal subarachnoid hemorrhage...


text:
- Jessa Duggar shares a rare photo of her brother Josh Duggar's daughter Meredith - US Weekly
- Michael Lohan's estranged wife Kate Major is detained by cops at an airport - TMZ
- Is there something between Ben Affleck and Halle Berry? - Gossip Cop
- The Hunger Games author Suzanne Collins says goodbye to the popular series after the final movie is released - Just Jared Jr
- What happened backstage with Kylie Jenner at The Ellen Show? - Radar
- Bryan Cranston tears up over a Breaking Bad memory - Huffington Post
- Leonardo DiCaprio's Oscar race heats up - Lainey Gossip
- The Captain America: Civil War movie may be better than the comic book - The Hollywood Reporter
06 January 2014 at 4:39 AM
Olivia Wilde & Jason Sudeikis: Snowy NYC Coffee Couple
About this photo set: Olivia Wilde is almost unrecognizable as she bundles up from the snow in a parka and sunglasses while out and about on Saturay afternoon (January 4) in New York… Read More Here
Photo #: 3023021
Posted to Jason Sudeikis, Olivia Wilde, Pregnant Celebrities


text:
Mini Commuter Messenger Bag | Performance Tweed
Overall rating based on 17 reviews
I've had this bag for a few days and it's nearly perfect for my needs to/from work and when I'm out and about. It is attractive and light and the magnet open/close is the best! It holds just enough of what I need to get around. Here are a few things I would improve on:
1. Put netting on the external side pockets so it can expand and hold a water bottle.
2. Make the bag a touch wider and perhaps a bit taller so we can fit in a folder or some papers without bending the edges. Right now, I have papers in there but it's just a bit cramped.
I love this bag and am looking forward to putting it through the ringer! Oh and the customer service at Rickshaw is amazing! I called to tell them was going out of town and they expediated my order so I had it to use in time. Thanks, Rickshaw!!
Jeffrey Kramer – Verified Buyer
Overall, a awesome bag at premium quality that i can highly recommend.
Bought a zero messenger medium, and a smaller one about a year ago, they still look as NEW, incredible.
But please, find a way if possible to lower shipping charges to NL.
Inga H. – Verified Buyer
I'm not certain you make my bag any more. One of my friends tried to order one like mine but could not find it on your website and settled for something very similar. I'm wondering if my bag was redesigned. In any case, I love mine, but hers is better!
Mine is more like a camera bag and hers is more like a purse. I use mine for a purse, so the lighter weight that hers is would be better for me.
For mine: It is a very nice looking bag, the sides do not collapse when I open it (big win!) I love the pockets. All zippers and fasteners are very sturdy. My only two complaints are: 1) it is heavy and 2) I have a fastener (like the kind that fastens a bicycle helmet beneath the chin) that I do not use and I wish were optional or removable. Overall the pros outweigh the cons by a huge amount. I wish I could split the fifth star in half. It is really a 4 1/2 :)
In any case, I love it and keep up the good work!
Neil Vincett – Verified Buyer
I absolutely love my new bag. Its very warm were we live in Summer and its always good to have water with me my new bag allows me to do that.My tablet fits in it perfectly . The bag does all that I want it to and is very handsome. One small thing, one of the clips on the strap has come loose a couple of times. I like to put the strap over my head and twice now the bag has nearly fallen. Its no big deal. The fact that it is made in San Francisco , one of my favorite cities, makes all the more appealing.


text:
Hot Gossip
Romance Report for Aug. 13
- Photo: Matt Sayles/Invision/AP3 of 6
'Twilight' Stud Gets Dumped by '90210' Hottie
Kellan Lutz may know his way around a push-up, but he apparently still has a thing or two to learn about romance. E! News reports the "Twilight" beefcake has been kicked to the curb by girlfriend AnnaLynne McCord. "She broke up with him," says a mole. "He wasn't a good boyfriend." In what specific areas was he lacking? Seems there was an incident over New Year's involving his lips and another girl, although the "90210" starlet "forgave him" for the alleged trespass, according to the snitch. But it appears she wasn't quite as understanding when he failed to produce a present for her birthday last month. "He showed up at her house with flowers and took her to dinner, but that was it," recalls the insider. "No gift." In Lutz's defense, it's not like he completely dropped the ball on her big day, and he reportedly tried to patch things up by booking a romantic retreat at a Santa Monica hotel, only to be told by a fed-up McCord that it was too little, too late.
Get out the vote
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text:
Working Class Formation in Taiwan
Fractured Solidarity in State-Owned Enterprises, 1945-2012
|Publication Date
|June 2014
|Hardcover Ebook (PDF) Ebook (EPUB)
|Palgrave Macmillan
Ming-sho Ho applies Marxist theoretical perspective to understand the postwar trajectory of Taiwan's state-sector workers. A global and comparative framework is used to examine the particularity of Taiwan's working class. It revises the stereotypical image of labor docility by showing ethnicity, party-state, and internal labor market produces intra-class divides and generates a variety of workers' resistance even under the repressive rule of one-party authoritarianism. The book looks at the rise of independent labor movement in the wake of political liberalization in the late 1980s. The similar current of social movement unionism of South Africa, South Korea and Brazil is also present in this oft-neglected case. Ho observes how labor activism gradually resides as democracy is consolidated and neo-liberalism becomes the new ideological hegemony.


text:
Synonym Center
- What is a synonym?
- What is an antonym?
- What is a thesaurus?
- What is an adjective?
- What is a verb?
- What is a noun?
- What is an adverb?
noun. fraud, trick
verb. set, dictate
adj. not clean mentally, physically; mixed
Roget's 21st Century Thesaurus, Third Edition Copyright © 2013 by the Philip Lief Group.Cite This Source


text:
Latest members
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text:
ChoiceStream, Inc.
|ChoiceStream, Inc. Patent applications
|Patent application number
|ACTIVE AUDIENCE CONTROL - An active control of advertising is enhanced through the use of solicited information to gain knowledge about a desired population of users. In some examples, this knowledge about users provides a self-identification of target users, and this self-identification is used to select further users with similar characteristics, who would also be likely to self-identify themselves as target users.
|RECOMMENDATION JITTER - A method for selection of items for one or more users includes performing each of a set of steps to determine the items. Performing the steps includes introducing a controlled variation into a result of each of multiple of the steps. The items for the one or more users are then selected according to the results of the steps.
|OPTIMIZATION OF A WEB-BASED RECOMMENDATION SYSTEM - A method for determining product recommendations to be presented to users includes forming, by a formula generation module, a plurality of different recommendation formulas, including, for each recommendation formula, assigning a weight to at least some of a plurality of recommendation characteristics, wherein each recommendation characteristic is representative of at least one of a characteristic of a product, a characteristic of a method for presenting the product recommendations to the users, and a characteristic of a user. The method further includes iteratively performing the steps of: for each of the plurality of recommendation formulas, selecting, by a product recommendation module, at least one product for presentation to the users on the basis of the corresponding recommendation formula; sending, by a communications module, instructions to a server to present the selected product to the users; receiving, by a data evaluation module, data representative of user responses to each of the products presented to the users; evaluating, by the data evaluation module, the received data; and selecting, using the data evaluation module, a subset of the recommendation formulas included in the plurality of recommendation formulas on the basis of the evaluation of the collected data.
|PERSONALIZED ADVERTISING AND RECOMMENDATION - Systems and methods for recommending items to users include maintaining a database of user-related information including user profiles, each including at least a history of user activities associated with a first entity that has a relationship with an inventory of recommendable items; obtaining information about an identity of a user interacting with a second entity different from the first entity and different from the service provider; associating the identity of the user interacting with the second entity with a corresponding user profile; selecting a first set of items from the inventory for presentation to the user based at least on an analysis of a history of user activities associated with the first entity; and forming a specification of the selected first set items for presentation to the user during the user's interaction with the second entity.
|STATISTICAL PERSONALIZED RECOMMENDATION SYSTEM - A method for recommending items in a domain to users, either individually or in groups, makes user of users' characteristics, their carefully elicited preferences, and a history of their ratings of the items are maintained in a database. Users are assigned to cohorts that are constructed such that significant between-cohort differences emerge in the distribution of preferences. Cohort-specific parameters and their precisions are computed using the database, which enable calculation of a risk-adjusted rating for any of the items by a typical non-specific user belonging to the cohort. Personalized modifications of the cohort parameters for individual users are computed using the individual-specific history of ratings and stated preferences. These personalized parameters enable calculation of a individual-specific risk-adjusted rating of any of the items relevant to the user. The method is also applicable to recommending items suitable to groups of joint users such a group of friends or a family. A related method can be used to discover users who share similar preferences. Similar users to a given user are identified based on the closeness of the statistically computed personal-preference parameters.
Patent applications by ChoiceStream, Inc.


text:
Christian Dior
The bother of the other: Dior, Louis Vuitton and Gucci Cruise along for 2016
You know what I like about the pre-collections? Not the travelling (I'm lazy), nor the dinners (I'm anti-social), definitely not the jet-lag, frequently not the clothes – they're often simple-minded, and, generally most aren't any good from a critical standpoint. What I like though is the time. The time to look at said clothes, to ponder them, frequently to turn them inside out. The fact you get to step off the increasingly frenzied fashion treadmill and actually spent a chunk of your day thinking about what you've seen, rather than rushing to the next.
By Alexander Fury | Fashion, Fashion Sense | Monday, 8 June 2015 at 6:15 pm
Dior and I: an artful argument for a new couture
It's really difficult to get fashion on film right – because it means so much to the people who love it, and is worthless to those who loathe it. Nothing will change those points of view – not extolling the hours of work poured into intricate clothes, not bandying about awe-inspiring figures about fashion house turnovers, not filming seamstresses weeping with emotion as their work is showcased, or possibly ripped to pieces. I personally fall into the former camp, so no matter how much you question the worth versus the cost or roll your eyes with puritan disgust at the vanity of it all, I won't be swayed.
By Alexander Fury | Fashion, Fashion Sense | Monday, 23 March 2015 at 7:22 pm
Junya Watanabe and Balenciaga round it out, in Paris
Context does a lot for fashion. I'm not just talking about the world outside the tent, or salon, or wherever designers have positioned their gilt chairs for this season, but about what other designers are showing in their salon/garage/specially-erected plexiglass cube (depends on budget). Of course, part of the dance of fashion is solipsism. Or maybe megalomania. It's rare you see designers at each others' shows: Tim Blanks and the team of style.com were trying to interview Joseph Altuzarra, attending the Balenciaga show designed by his friend Alexander Wang, but were constantly interrupted by well-wishers. Most said something along the lines of "I didn't expect to see you here!"
By Alexander Fury | Fashion, Fashion Sense | Saturday, 7 March 2015 at 12:57 pm
"Viva la Mamma." Every day is mother's day, for Dolce and Gabbana
It isn't Mothering Sunday until May in Italy – at least, not officially. But Domenico Dolce and Stefano Gabbana care naught for that kind of stuff. The Sunday of every Milanese womenswear week is traditionally Dolce day, but this time they made it mother's day, too. "Viva La Mamma" they called their show – in English, it translates oddly to "Hurray for Mum" – less Italian romance, more the post-war jolly hockey sticks Enid Blyton school of British children's literature.
By Alexander Fury | Fashion, Fashion Sense | Sunday, 1 March 2015 at 3:36 pm
Raf Simons' Dior esprit – don't call it 'pre' – in Tokyo
Raf Simons' latest Dior collection marched out across the square dais of Tokyo's Ryōgoku sumo hall – an arena more akin to fat men slogging it out in underpants than thin women trying to sell us expensive ones. This is Dior's contribution to a season most of the world dubs pre-Fall – the no-man's season that lies between spring/summer and autumn/winter.
How long will fashion journalists feel the need to explain that? Possibly a good while longer if houses continue to come up with inventive names to mask the issue they all seem to have with "pre".
By Alexander Fury | Fashion, Fashion Sense | Thursday, 11 December 2014 at 6:33 pm
Standing to attention: at Raf Simons, clothes worth a closer look
The coupling of great clothes and a great show, where neither one outshines the other, is very rare. It's all the more remarkable, then, that Raf Simons' formula for his eponymous label seems such a sure-fire hit. I tried to think back to a Simons show that hasn't worked, gloriously. Then I realised this was his business' twentieth anniversary. I couldn't think of a single instance.
By Alexander Fury | Fashion, Fashion Sense | Thursday, 26 June 2014 at 1:23 am
Oil and Water: Chanel, Dubai, and the retail phenomenon of "Cruise"
There's a trend right now for fashion houses to show the collections we still dub Cruise in far-flung locales. I'm writing this in the airport in Dubai, following Chanel's show; a week ago Dior Cruise-ed to New York and chugged us from Manhattan to Brooklyn on a chic branded ferry; last summer, that house showed in Monaco, coincidentally the site of Louis Vuitton's inaugural Cruise show this weekend.
By Alexander Fury | Fashion, Fashion Sense | Tuesday, 13 May 2014 at 10:04 pm
Cruise 2015: Plain sailing from Raf Simons' Dior
It's odd seeing a Dior show in New York. Namely, because it all feels so very Parisian. Actually, it's Parisian per se – which conjures up images of chi chi little pied-de-poule suits and veiled hats, baguettes, breton stripes, clipped poodles, that ooh-la-la Francophile shtick – but reminiscent of the Dior we see in Paris. There was the same stripped-back set, this time an expanse of white catwalk and a metallic mirrored backdrop reflecting the scene like an idealised Hudson River. We were watching Cruise, after all. The real Hudson was more Dior grey than mirror, in reality, but even that fitted the identity of the house.
By Alexander Fury | Fashion, Fashion Sense | Thursday, 8 May 2014 at 2:47 pm
The "Off season": Cruising, Youtubing and the interesting boredom of Dior and Louis Vuitton
I bet people wonder what fashion editors do during the "off season": write dodgy novels, maybe? Shop? Painstakingly plan ensembles to be crated and shipped ready for the next round of shows, when said eds are released from their pen, like the rabid hounds that shred unwanted visitors to Mr Burns' mansion in The Simpsons? Only fashion editors voraciously attack and devour garments, and sometimes designers, rather than intruders.
By Alexander Fury | Fashion, Fashion Sense | Friday, 25 April 2014 at 9:49 pm
Parisian provocations: At Dior, Raf Simons suits
We think of Dior as a house built on evening wear – the French call that flou, which, as the name implies, denotes garments with a sense of fluidity and lightness. Even if a Dior ball gown was anything else. But Dior's most famous fashion image is of la Tailleur Bar. It's one of those house codes that Raf Simons has been fixated on since he began redesigning Dior in 2012. Hence the fact the Bar suit's jacket, the nip-waisted, thrust-hipped silhouette originally cut in tussore silk by Pierre Cardin (a tailor at Dior before setting up under his own steam) appears again and again, insistently, in his collections, cut in everything from grain de poudre to denim.
By Alexander Fury | Fashion, Fashion Sense | Sunday, 2 March 2014 at 2:43 pm
Latest from Independent journalists on Twitter


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Bunyip Tours Managing Director George Josevski was recently searching Google Images seeking fresh images of the Great Ocean Road for business promotion activities and he discovered the photography of award-winning Australian photographer Darren Stones.
"George said to me on the phone that he was impressed with my photography and that he wanted to license the use of five of my images," said Darren. "Whilst on the phone, George invited me to inspect the Bunyip Tours website and I later advised him on the style of imagery that could enhance his website, brochures and posters. He generally uses images supplied by Tourism Victoria, but he was after something new, fresh and different. I was able to help him."
Darren Stones Visual Communications is currently supported by the Australian Government as part of the New Enterprise Incentive Scheme (NEIS). The program provider is the Greater Melbourne Employment and Training Services and is delivered at the Kangan Institute of TAFE in Essendon, Victoria.
# # #
Darren Stones Visual Communications, provides photographic services and merchandise and writing services for improving the visual communications of travel, tourism, corporate, publishing and retail businesses.


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Hats off to Petrucci, again showing well on the Ioda. Sacchi obviously saw something there, I know its nearly 3 seconds back but the CRT's will gain more than the factory bikes and never expected anything from him.


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Remember Me
Leftover Salmon isn't just a band they are an experience. Call it what you want - new grass, progressive bluegrass, slam grass, "polyethnic cajun slamgrass" or even just the best time you can have in bare feet. Leftover Salmon has been called all those things and more. Years in Your Ears is a documentary that captures a time not long ago when these forefathers of today's "jam-grass" scene roamed the earth, promoting their festival attitude, inspiring groups like The String Cheese Incident and Yonder Mountain String Band, and helping make the Colorado music scene what it is today.
The DVD explores the attitudes and influences that made Salmon what they are. It examines the controlled chaos that was and is Leftover Salmon, and their connection to both bluegrass music and their fans. Time and time again, Vince Herman and Mark Vann held audiences in the palm of their hands. The DVD does a great job weaving anecdotes about the band with clips of Salmon in action from their earliest days to their glorious end run. Leftover's signature jams accompany some amazing still photos, including a touching one of a young John Bell and Mikey Houser (Widespread) jamming with them.
The interviews are plentiful and insightful, and include SCI's Michael Kang and Bill Nershi, Yonder's Jeff Austin, Dave Johnston and Ben Kaufman, as well Sam Bush and Little Feat's Bill Payne and Paul Barrere. Music highlights include: "Pasta On the Mountain," "Carnival Time," "Voodoo Queen Marie," "Dance On Your Head," "Bend in the River" and an epic "Gonna Have A Party" montage.
Banjo player Mark Vann's passing is discussed in a beautiful way, shining a light on the joy his life and playing brought to all those around him. I was anxious to see how Years in Your Ears would approach this delicate subject, and was delighted they did so with such humor and class.
Bonus features include rare music tracks and interviews with Vince Herman and Drew Emmitt. Thanks in large part to the lower energy required to run digital equipment and a personal dedication to the environment, producer Eric Abramson was able craft this DVD from largely renewable energy resources including wind and solar energy - a personal dedication to the planet that should make any Salmon head proud.
This DVD hits the market just in time for fans wishing to reacquaint themselves with Leftover before the summer concert season is upon us. This year will be a hallmark year for Leftover Salmon fans as it marks their return to the stage when it seemed unclear if this would ever happen again. Their shows are some of the most memorable fun one can have and this DVD captures that glorious feeling eloquently.
To purchase Years in Your Ears click here.
JamBase | Colorado
Go See Live Music!


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How to Brew Tea
Tea is a simple pleasure. Here are some simple, authoritative tips on how to make a cup of tea. 1. Buy good tea, and keep it well A good cup of tea begins with your tea merchant. You can ruin … Read More
Local singer/songwriter Jerry Williams to perform at Portsmouth Tea Sessioons
18-year old Jerry Williams, from Milton, is fast becoming Portsmouth's brightest up-and-coming star of the music scene. She won Runner up for Best Solo Artist for the Portsmouth Guide Awards 2013. She released her first EP, A Hairdresser's Called Sids, … Read More
Retailer of the Year 2013
We won the Retailer of the Year Award at the News Business Excellence Awards last night.
Should you put the milk in first or last?
Here's a question I'm asked several times a day. I don't mind answering it, but I thought it might save everyone time if I wrote the answer down. Should you put the milk in first or last?
Comical Musician Arnold Gutbucket loved Portsmouth Tea so much that he wrote a song about it
Local musician Arnold Gutbucket – also known as James Birmingham – has two great loves: music and Portsmouth Tea. Now, he has combined his two passions: by writing a song about the popular local brew. He premièred the song at … Read More


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Latest Weather Blog
No tenure for new school bus drivers
BATON ROUGE- New Louisiana school bus drivers will no longer be able to reach the job protection status called tenure, under a bill given final passage Monday.
Currently, school bus drivers have a probationary term of three years. If they are not dismissed or found unsatisfactory during that time, they reach tenure, which gives them a permanent job status and makes it harder for them to be fired.
Louisiana is in the only state in the nation that has tenure for bus drivers.
The measure (House Bill 293) by Rep. Joe Harrison, R-Napoleonville, will remove tenure for any bus drivers hired after July 1. It won't take away tenure for bus drivers who already have it.
The House voted 67-30 for the measure, and the Senate voted 20-15. Approval sent the bill to the governor's desk.


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Scooter Shop Gold Coast | Scooters Gold Coast | Aprilia Scooters | SYM Scooters | PGO Scooters | New & Used Scooters
The Scooter Shop, the place to buy and service scooters on the Gold Coast. Aprilia, SYM, TGB and PGO. Servicing and repairs for scooters on the Gold Coast.


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2000 Buick Century • 150,000 miles
I took my car to an auto part store to have it plugged in to see what was wrong with it. They said I needed a new control valve and a mass air flow sensor. I bought and had both of those changed. No other codes showed up besides those ones. My car doesn't idle as bad as it did before we changed these two parts but it is still idling very high when you are stopped or slow down. It sometimes dies. We also put a can of fuel injection cleaner in the gas tank. Nothing seems to be working with it idling so high and dying. It also gives off a funny smell when it idles sometimes. The smell doesnt happen everytime it idles but does sometimes. I need help with getting this fixed!
July 13, 2012.
July 13, 2012.
What were the exact code numbers you had?Did you clean the port where the idle air control valve goes in before replacing it?Also sounds like they justed guessed and sold you lots of nice new parts. Most of parts counter people have never even worked on a car before. Have you looked for vacuum leaks?
Jul 13, 2012.
Jul 13, 2012.
A couple months ago my vehicle started to produce a gasoline smell whenever I had the air conditioning/heat on idling at a stop sign/light or in park. Two different mechanics that I ...
2 answers • 2004 Jeep Grand Cherokee • 115,000 miles
When I was driving home from work today I noticed a burnt rubber or plastic smell in the inside of my car. When the heat is on or off, the smell is still there. I checked the hood for...
3 answers • 1995 Mazda Protege • 148,000 miles
While driving my car from store to store I started smelling burning plastic, I had my air on max and decided to turn it off, but could smell it burning, so I parked the car for a few ...
2 answers • 2004 Ford Taurus • 101,790 miles
I have a 2005 dodge magnum rt that has a smell like paint thinner on the outside and inside coming thru the vents. This is a recent problem.
11 answers • 2005 Dodge Magnum • 77,000 miles


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What travellers are saying about this Hotel
Are you the business owner?
9 traveller reviews for Clarks Serviced ApartmentWRITE A REVIEW
- Arun Sharma
- Lives in Pune
- 2 Reviews
"Awesome appartments for staying"
- Posted May 24, 2015
I travelled to Goa with my friends in the month of Feb 2014, and we booked multiple 2BHK and 3BHK rooms for our stay as we're 30+ peoples. All the rooms were very clean and were having the all the amenities like sofas, fridge, LCD TV and water purifier. All the facilities were up-to the mark. All the rooms and halls are very much spacious. However 3BHK rooms were having much more good furniture and bigger LCD TV. There was also a common swimming pool which we can use. Only cons I can say is that for one 2BHK room (There are two kind of 2BHK rooms, depends which kind of room you get), it is having only one common bathroom. So the persons staying in it has to wait till it is occupied. Apart from this I can not see any other issues in the rooms. As we spent most of the time roaming outside and on the beaches so we never get the time to use the room service. Though we had to remind them to provide the fresh towels next day. Also you have to walk around 1-2 Km to get to the beach, but it was fine with us as we walked through the Calangute market.More » « Less
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- Arathi Gannavarpu
- Lives in Hyderabad
- 1 Review
"Good room with amenities; hygienic food"
- Posted Nov 4, 2014
The apartment was conveniently located. We did not find any difficulty in reaching there. The Staff behaviour was good and service provided by them was nice. They were quick to attend all requests of the customer. The appearance of the hotel is good. Room was good. The bed sheet was not hygienic. Amenities like AC, TV and a geyser were in a working condition. We had food here only. The food was tasty and hygienic. We did not face any delay during check-in and check-out. There was no garden at the hotel. The hotel is reasonable.More » « Less
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- Divin T c
- Lives in Agra
- 2 Reviews
"The apartment is conveniently located"
- Posted Oct 29, 2014
The apartment was conveniently located. The appearance of the apartment was attractive and hygienic. Calangute beach was one and a half kilometre from the apartment. The staff was professional and hygienic. The rooms were well furnished and hygienic. There was some problem in check-in and check-out procedure, but later on solved by the management. Kitchen, bathroom was hygienic. There was no view from room. The food they don't provide. The parking facility was there. The place was fully secured by securities. Wi-Fi facility was there. Its valued for money.More » « Less
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- Parth Gautam
- Lives in Bangalore
- 1 Review
"The staff at the hotel were helpful and friendly"
- Posted Sep 30, 2014
The appearance of the hotel was quite good, well-maintained. There was no delay while check-in and check-out procedure. The staff at the hotel were helpful and friendly. Rooms were spacious, clean, neat and well-maintained. All the basic amenities were functioning properly. There was no view from the room. I did not taste the food in the hotel. There were no games or any other activities. Overall, I was happy with the stay and I would like to recommend to other travellers.More » « Less
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- Ali Khan
- Lives in Lucknow
- 5 Reviews
"Good for family stay with good service & food"
- Posted Sep 22, 2014
I would highly recommend clarks service apartment for family stay, we had a wonderful experience. The place, the service and the staff has built a special memory for us, their hospitality, food, personal attention towards everything is remarkable, they actually treated us like family guests and not just strangers staying in a hotel. Mr. Varma, Adil, Sanju everyone on the staff thank you so much for the wonderful time...More » « Less
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- Amol
- Lives in Pune
- 1 Review
"Nice interior and facility"
- Posted Dec 6, 2013
Hotel is situated near calangute beach, which give a very nice experience. You can walk through the markets any time. Hotel is very good, it has very good interior and the house is very clean. The service is OK. Very nice hotel when you visit with more family members. We were 11 members and the apartment was enough to accommodate all of us easily.More » « Less
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- Rkane K
- Lives in Mumbai
- 1 Review
"They were rude & no courtesy at all"
- Posted Sep 23, 2013
I would like to inform/alert that we group of 10 loved the place when we went thru the website. But the kind of time we spent on calling/mailing the reservations team in Delhi was too much. We transferred the advance and we got a confirmation on mail, after 5 days we got a mail stating 'we cannot allot any rooms for you". It was the first time in my life when such a thing happened. They were rude & no courtesy at all. Imagine this happening to you before 10 days of travel Finally we had to search for options just before the travel. Hence be careful.More » « Less
- Was this review useful?
- 1 vote
- Rajesh Patil
- Lives in Mumbai
- 1 Review
"kitchen facility provided is good."
- Posted Jun 12, 2013
Had a very good stay at Clark\'s apartment. They have got excellent rooms with good service. Swimming pool is also clean and quite big. The kitchen facility provided is good. The overall ambiance of the apartment is pleasant. It is also near to calangute beach.More » « Less
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- Yash Gupta
- Lives in Delhi
- 1 Review
"A very good experience"
- Posted May 8, 2013
Rooms wese very spacious . .and every day neded things were already there in the apartment. Overall it was a very good experienceMore » « Less
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Have you stayed at Clarks Serviced Apartment?
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Calangute hotels travelers are looking right now220 Reviews101 Reviews103 Reviews17 Reviews
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There are 9 traveller reviews for Clarks Serviced Apartment. The minimum tariff for Clarks Serviced Apartment is Rs.3,350 and the maximum rate charged is Rs.3,350.


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to wonder why we, and our mums, used to wear the silly garments...(116 Posts)
KatrineEM Mon 20-May-13 20:25:06
MrsHoarder Tue 21-May-13 12:05:20
TheSmallClanger Tue 21-May-13 12:10:32
Chandon Tue 21-May-13 12:15:43
frillyflower Tue 21-May-13 14:50:06
YoniTime Tue 21-May-13 22:42:09
garlicgrump Tue 21-May-13 22:51:51
garlicgrump Tue 21-May-13 22:54:30
PiratePanda Tue 21-May-13 23:01:41
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burberryqueen Tue 21-May-13 23:11:44
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nickschick Tue 21-May-13 23:30:51
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August 13, 2011
Welcome to Moms Of New Braunfels Blog
It is also an extention of the Moms of New Braunfels Facebook Group
Moms of New Braunfels on Facebook
This group is open to ALL moms who live in and around New Braunfels, regardless of whether you work or stay-at-home and regardless of the age of your children.
4 comments:
I was looking up Fun Places for Kids to take my son in New Braunfels, do you have any suggestions?ReplyDelete
- Anonymous4/10/13 9:35 PM
Please do not help the puppy mills by offering your yorkie or boxer dog to them....ask them to consider adoption from a shelter....they don't even know how to spell breeding in the ads!! [breading] (sic) stop the insanity and madness.....administrator please strike this ad immediately!!!ReplyDelete
- Anonymous27/9/14 12:25 PM
hi this is DJ Marco from River rat DJ service. I would like to think mothers of New Braunfels for suggesting my business for the Reynolds wedding. I received the job and th that you suggested me I appreciate all of your help thanks again DJ Marco 21386 48044ReplyDelete
- Anonymous9/3/15 1:22 PM
Mommy's (and daddy's) read....if you dare. I did.ReplyDelete


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This blog is owned and operated by the ANZ ConfigMgr Premier Field Engineer team.
Ian BartlettMatt ShadboltGeorge Smpyrakis
Blog Links
My last post on ConfigMgr 2012 Collections (I promise!). I wanted to go over Include and Exclude Collection Rules.
Include and Exclude collection rules are a new feature in ConfigMgr 2012 and I think you'll find them most useful. They basically allow us to add or remove resources from a collection without having to write a complex membership rule.
Let's say we have five collections: Finance Accountants, Finance Banking, Finance Bookkeeping, Finance Insurance and Finance Interns. Each of these collections will have their own Deployments targeted at the users in the collections. If we wanted to target an application at all of these collections, we would need to have 5 deployments.
Or we could create a collection with an Include Collection setting
Now we can just target the application at the Finance – All collection and we'll get all three members.
Exclude collections are even cooler. Let's say we have a collection for all computers who need Office 2010. Normally we would just deploy our application to the collection and hope for the best. We can use an Exclude Collections rule to be a little smarter in our deployments and remove all clients who are in an 'unhealthy' state.
First, we'll create our Unhealthy Clients Collection. Our membership rule will be any looking for any client who's client status is NULL or the client activity is NULL. This should get any computer that hasn't got the client installed, or any computer with the client installed that isn't active. (this is a *really* basic query for unhealthy clients. You can write a more complex one to find those clients who are truly unhealthy)
I've got 4 systems in my lab hierarchy – only one of which is healthy (oh, and the two Unknown Computer objects – but they don't count)
So when I update the membership of my Office 2010 membership, I will only get the healthy client.
Using this exclusion method has a few benefits – firstly, you have a collection full of systems in which you have to repair the ConfigMgr client. Secondly, you're software deployment statistics should be a lot cleaner as you're not getting failures on the unhealthy clients. Finally, as the collection is dynamic, as soon as you remediate the unhealthy clients, they will receive the Office 2012 deployment!
Matt Shadbolt
Hi Matt
Thanks for the post - just a minor thing which doesn't affect the content of the posting, but noticed you said about the Office 2010 membership, then in the image you have Install Office 2012, and the last paragraph has Office 2012 listed too.


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The Women's Chakra Bodyfit 200 Zip Top by Icebreaker is a raglan long-sleeved top has a high neck and a versatile zip. You control the temperature. The seams are designed flat for comfort. This is a classic style that you will use all day long, night long and year long.
So sorry, but only 4 items are allowed for comparison. I blame the gnomes and so should you.
Would you like to compare the products you have selected now?


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Ole Miss and Vanderbilt will not only play their 2014 football game at an NFL stadium, but it will also be nationally televised by ESPN. Kickoff for the teams' Sept. 6 SEC opener will be 3:30 p.m. CT.
Click here for the original story.
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There are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for β-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter.
Gladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5′ GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5′UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5′UTR-intron. The 5′-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis.
The GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5′UTR-intron from GUBQ1 resulted in a similar pattern of β-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.
There are many non-cereal monocots of agronomic (garlic, onions, banana, sugarcane, millet), horticultural (turf and forage grasses such as tall fescue, Kentucky bluegrass, creeping bentgrass, perennial ryegrass, St. Augustinegrass, buffalograss, Zoysiagrass japonica, Paspalum vaginatum, Paspalum notatum, floral crops such as orchids, lilies, tulips, crocus, amaryllis, iris), and potential biofuel (switchgrass, Miscanthus) importance. Successful transformation of these species requires an understanding of how their gene expression is controlled by promoter elements such as introns.
Introns have been found to significantly stimulate gene expression. Dicot introns have been used to transform dicot plants, and levels of intron-mediated expression (IME) in leaves ranged from 2–20× [1-4]. Monocot introns have been shown to enhance gene expression in monocots 20–100× [5-10]. Most of the studies with monocots have been transient transformation studies using callus cells or protoplasts of cereal monocots rather than stable transformation of plants to characterize expression within specific plant tissues. There have been a few transient transformation studies showing IME of non-cereal monocots (banana, lily, bluegrass) using introns (maize Ubi1Arabidopsis UBQ) isolated from either cereal monocots or dicots [11-14]. Half of the introns tested by Vain et al. [12] showed different results on transient expression for maize and bluegrass indicating a difference between cereal and non-cereal monocots.
This is the first study using an intron isolated from the promoter of a non-cereal monocot and evaluating its effect on expression in both a dicot and a monocot plant. Previously we had reported that the non-cereal intron from the GUBQ1 promoter from Gladiolus enhanced GUS expression in suspension cells [15]. It is critical to verify IME using transformed plants as IME has been shown to differ dramatically depending on the tissue it is expressed in [4,16,17]. The sugarcane ubiquitin promoter including its intron expressed GUS at high levels in sugarcane callus, but there was no expression in plants of sugarcane [18]. Results in this study show that IME occurs in both a dicot and non-cereal monocot plant with the GUBQ1 intron. This information will contribute to our understanding of factors controlling gene expression in a non-cereal monocot and can be used when genetic engineering applications require high levels of expression.
Southern hybridization
Gladiolus and Arabidopsis plants were transformed with the uidA gene under control of either a 1.9 kb GUBQ1 promoter (G1-1), a 5′end of GUBQ1 that is lacking the intron (G1-3), or the CaMV 35 S promoter (Figure 1). Southern hybridization confirmed integration of the uidA gene in Gladiolus plants (Figure 2A). Hind III used for digesting the genomic DNA of Gladiolus and Arabidopsis cuts once immediately 5′ of the GUBQ1 or CaMV 35 S promoter region. Three plants of Gladiolus that were shown by the Southern to be independently transformed had 2–5 copies of the uidA gene under control of G1-1. Gladiolus plant lines transformed with the uidA gene under control of either G1-3 or CaMV 35 S were also multicopy with 4–7 and 3–7 copies, respectively, of the transgene.
Figure 1. A. Diagram of the DNA constructs used for transformation.Gladiolus GUBQ1promoter (G1-1) and an intronless version (G1-3). TS, transcription start; E, 5′UTR-exon; 5′U-I, 5′UTR intron; 35 S, CaMV 35 S promoter. Negative numbers represent the nucleotides on the 5′ side of the translation start codon. For Gladiolus transformation pUC-GUS was used as the backbone vector, pCAMBIA1391Z for Arabidopsis transformation, and pBI121-GFP for transient transformation of tobacco. B. Relative fold increase in the specific GUS activity for leaves from Arabidopsis (white bars) and Gladiolus plants (shaded bars) transformed with either the CaMV 35 S, G1-1, or G1-3 constructs. Three independently transformed lines were used for each of the constructs analyzed for GUS expression. Standard error shown.
Figure 2. DNA blots of genomic DNA isolated from (A)Gladiolusand (B)Arabidopsisplants containing theuidAgene under control of either the CaMV 35 S (35 S), G1-1, or G1-3 promoter. Genomic is digested with Hind III and hybridized with a 511 bp uidA specific probe. Numbers indicate the transgenic plant lines or non-transformed (NT) plant. Two size markers, 3 and 10 kb, are shown.
Approximately 30% of the transformed Arabidopsis plants contained a single copy of the uidA gene, and only those with a single copy of the uidA gene were used in this study (Figure 2B).
GUS expression
Histochemical staining showed GUS expression throughout the leaves of transgenic Gladiolus plants with the CaMV 35 S and G1-1 promoters (Figure 3A). High levels of specific GUS activity were comparable with the GUBQ1 and CaMV 35 S promoters in leaves of Gladiolus from plants transformed with either the G1-1 or 35 S promoters (Figure 1B). Specific GUS activity measured in leaves of transgenic Gladiolus plants with G1-3 was significantly lower than that of G1-1 plants indicating the importance of the intron on expression (Figure 1B). GUS expression was not evident from histochemical staining in leaves and roots of Gladiolus plants transformed with the G1-3 promoter, and light blue staining indicative of GUS expression was present in callus found at the basal meristem region of G1-3 plants (Figure 3A). A quantitative comparison of GUS activity in leaf, root, and callus confirmed the low level of GUS expression in both types of organs and callus tissue with the G1-3 promoter and the high level of expression with the G1-1 promoter (Figure 4).
Figure 3. Histochemical GUS staining of transgenic (A)Gladiolusand (B)Arabidopsisplants containing theuidAgene under control of either the CaMV 35 S (35 S), G1-1 or G1-3 promoter. The Gladiolus plant material was taken three weeks after subculture from plantlets growing in vitro on MS medium. Plant tissue was incubated 16 h at 37°C in the staining solution. Magnification bars in A represent 2 mm.
Figure 4. Expression of theuidAgene in transgenicGladiolusorgans and callus. GUS specific activity was measured using three plants for each of the three transformed lines, and the mean and standard error are shown.
Arabidopsis showed higher levels of specific GUS activity with the GUBQ1 promoter than the CaMV 35 S promoter (Figure 1B). Histochemical staining showed GUS expression throughout the entire leaves and roots of transgenic Arabidopsis plants with the CaMV 35 S and G1-1 promoters (Figure 3B). Staining occurred at the periphery in some of the leaves of Arabidopsis that had the G1-1 and G1-3 promoters. The blue staining was considerably lighter in Arabidopsis transformed with the G1-3 promoter as compared to the dark blue staining seen in G1-1 plants. The specific GUS activity was negligible in leaves with the G1-3 promoter confirming the importance of the GUBQ1 intron to expression in Arabidopsis (Figure 1B).
RNA and translation efficiency
The two constructs, G1-1 and its intronless version G1-3, produce identical mRNAs that have the same nucleotide sequence in the 5′ leader. In leaves of transgenic Gladiolus plants the level of uidA mRNA was two-fold higher with the promoter only and its 5′UTR (G1-3) as compared to the 1.9 kb promoter that included the intron (G1-1) (Figure 5A). In comparison, Arabidopsis leaves transformed with G1-1 had significantly higher levels of uidA mRNA than leaves with G1-3 (Figure 6A). The intron affected the level of uidA mRNA in Arabidopsis but not in Gladiolus. Splicing of the intron appeared to be occur efficiently from the GUBQ1 mRNA in leaves of Gladiolus. The trend in translation efficiency was similar for the two species (Figures 5C and 6C). In both Gladiolus and Arabidopsis the highest translation efficiency was with the 1.9 kb promoter (G1-1), and efficiency was negligible with the promoter and 5′UTR (G1-3).
Figure 5. Expression of theuidAgene in transgenicGladiolusleaves. A. Relative uidA mRNA levels. B. Specific GUS enzyme activity. Each bar represents a transformed plant line in A and B. Lines from left to right-G1-1: 1, 2, 3 and G1-3: 4, 5, 6. The black bar represents the average of the plant lines for each construct. GUS specific activity and uidA mRNA levels were measured using three plants for each line. The mean and standard error are shown. C. Specific GUS enzyme activity/unit uidA mRNA. Average GUS activity was divided by its corresponding mRNA level for each DNA construct.
Figure 6. Expression of theuidAgene in transgenicArabidopsisleaves. A. Relative uidA mRNA levels. B. Specific GUS enzyme activity. Each bar represents a transformed plant line in A and B. Lines from left to right-G1-1: 113, 117, 118, G1-3: 131, 132, 134, 35 S: 1, 2, 7. The black bars represent the average of the plant lines for each construct. GUS specific activity and uidA mRNA levels were measured using three plants for each line, and the mean and standard error are shown. C. Specific GUS enzyme activity/unit uidA mRNA. Average GUS activity of each construct was divided by its corresponding mRNA level for each DNA construct.
The act7 intron isolated from Arabidopsis was substituted with the Gladiolus intron in the G1-1 construct to determine the effect of a dicot intron rather than a non-cereal monocot intron on transient GFP expression and translation in tobacco. Although the Arabidopsis act7 intron did not increase GFP expression to the same extent as the Gladiolus intron, expression with the act7 intron was approximately half the level of G1-1 (Figure 7B), and there was a corresponding increase in GFP mRNA level (Figure 7A). The translation efficiency of the GUBQ1 promoter was higher when either intron, the Arabidopsis act7 or Gladiolus GUBQ1 intron were present (Figure 7C). The level of GFP protein produced in tobacco was found to be significantly higher with the Gladiolus GUBQ1 intron as compared to the act7 intron (Figure 8C bottom panel).
Figure 7. Transient expression of thegfpgene in tobacco leaves. A. Relative GFP mRNA level. B. Relative amount of GFP expressed. C. Specific GFP amount/unit mRNA of GFP. Average GFP level of three leaves for each construct divided by its corresponding mRNA level. G1-1(A) contained the Arabidopsis actin7 gene and its 5′-UTR intron (540 bp) instead of the Gladiolus 5′-UTR intron.
Figure 8. RT-PCR analysis using the primers 5′UTR-exon F anduidA200 R/gfpR in (A) transgenicGladiolusleaves (B) transgenicArabidopsisleaves, and (C) transiently transformed tobacco leaves. Western blot analysis of GFP shown in bottom panel of C. The vectors were pUC-GUS for Gladiolus, pCAMBIA for Arabidopsis, and pBI121 for tobacco. pCAMBIA contains "LacZ alpha, truncated" between the GUBQ1promoter and uidA gene". Abbreviations: Non-transformed plants (N), G1-1 (1), G1-3 (3), G1-1 promoter with act7 intron [1(A)], transient expression with pBI121 vector (−). Actin gene primers specific to each plant species analyzed were used as the reference gene to verify RT-PCR performance and the possibility of DNA contamination (bottom panel in A and B, middle panel in C).
It appears that in tobacco the Arabidopsis act7 intron was not spliced as efficiently as the Gladiolus GUBQ1 intron (Figure 8C top panel). Two bands were produced after RT-PCR with 1(A) primers for amplification of the Arabidopsis act7 intron (Figure 8C top panel). The 365 bp band is expected when the act7 intron is spliced out, and the second 910 bp band indicates that 1(A) splicing was incomplete. An actin gene specific to each species analyzed was used as a reference gene to verify RT-PCR performance and the possibility of DNA contamination (Figure 8A and B lower panel, C middle panel). RNA samples were not contaminated with DNA because only one band was present for actin. A second actin band would have resulted from DNA that did not have its actin intron spliced out (Figure 8A and B lower panel, C middle panel).
GUS expression in Gladiolus and Arabidopsis was dependent on the presence of the GUBQ1 intron. Sequencing of this intron showed that its 68% AU content is more similar to that of dicots that contain a minimum of 60% AU and an average of 74% AU in comparison to cereal monocot introns that average 59% AU, and this may explain why the intron functioned well in Arabidopsis and tobacco. Splicing of introns is more precise in dicots than in cereal monocots, and the AT nucleotides are thought to facilitate splicing in dicots [19]. More recently Rose et al. [20] examined intron sequences in the Arabidopsis thaliana genome and concluded that sequences that enhance gene expression are dispersed throughout an intron, and these specific sequences are conserved between monocots and dicots. Deletion of the intron from three polyubiquitin promoters, UBQ3UBQ10, and UBQ11 isolated from Arabidopsis, resulted in a 2.5–3× decrease in gene expression in Arabidopsis leaves [2]. Omission of the intron from the GUBQ1 promoter resulted in a much more dramatic effect, no expression, in Arabidopsis leaves as compared to its omission from the Arabidopsis-derived polyubiquitin promoters.
The role of the intron in controlling gene expression is typically more critical in cereal monocots than dicots, and it appears to be critical in leaves of this non-cereal monocot, Gladiolus. Levels of GUS expression were reduced to background in rice when the promoter's intron was absent from the rice Act1 and RUBQ2 promoters [21,22].
Studies with other non-cereal monocots have found that some, but not all, introns derived from cereal monocots may stimulate gene expression, and dicot-derived introns result in relatively lower levels of IME. A transient transformation study using lily and tulip leaves showed a decrease in GUS expression when the maize Adh1 intron was included with the CaMV 35 S promoter, and this same result occurred in sugarcane [11,23]. Chloramphenical acetyltransferase expression increased in protoplasts of Panicum and Pennisetum 23–28× following transient transformation with the CaMV 35 S promoter and first intron of the maize shrunken-1 locus [13]. This same intron resulted in higher, 91×, CAT expression in maize protoplasts. The maize Wx, maize Bz, rice Act1Adh1SShPetunia Chs, and maize Ubi1 introns were tested in bluegrass suspension cells using transient transformation, and the highest level of GUS expression, a 26X increase, was with the maize Ubi intron [12]. Transient expression using embryogenic cells of banana was enhanced 100×, 300X, and 10× when the maize Ubi1, rice Act1, and sugarcane RbcS introns were included with the banana bunchy top virus DNA-6 promoter, respectively [14]. The maize Ubi1 intron with banana bunchy top virus DNA 6 promoter was used for stable transformation, and banana plants had levels of GUS expression similar to that of the CaMV 35 S promoter [14]. It is difficult to predict the level of IME for each non-cereal monocot species and a particular intron. The maize Ubi1 and rice Act1 promoters showed very low levels of transient GUS expression in Gladiolus[24] possibly because of their introns, yet these two introns showed high levels of IME, 100–300×, in banana and only 26 × in bluegrass.
Rose and Beliakoff [25] have shown IME using either of the first two PAT1 introns. Because the levels of uidA mRNA increased during IME, but the rate of transcription remained constant, IME with the PAT1 introns was concluded to occur through a post-transcription mechanism. In a later study using Arabidopsis, the position of the tryptophan biosynthesis 1 (Trp1) and UBQ10 intron was varied in the coding and 3'UTR regions [26]. Both introns at all locations stimulated GUS expression more than mRNA accumulation. Results from this study support a model for introns operating at both the level of transcription and post-transcription. Rose's recent study of IME [27] using hybrid introns in either their normal orientation or reverse complement resulted in a model of IME that acts at the level of the transcribed DNA rather than the nascent RNA. Samadder et al. [28] found that IME with the 5′UTR intron of the rice Rubi3 gene increased the gene's mRNA level 20X and stimulated the intron's transcription 2X in a nuclear run-on experiment. Their results support both transcriptional and post-transcriptional effects of the rice Rubi3 intron, and suggest that the main effect was at the level of post-transcription. In both Gladiolus and Arabidopsis the 5′UTR-intron stimulated translation efficiency supporting the effect of the 5′UTR-intron at the post-transcriptional level.
In mammalian cells, genes containing an intron generally show more expression than the same genes lacking an intron. Wiegand et al. [29] did experiments to separate the effects of intron removal and exon junction complex formation on gene expression and found that the components of the exon junction complex function to activate splicing in human cells. Removal of an intron will not increase gene expression without exon junction complex formation. An increased amount of protein translated in mammalian cells was associated with splicing of the protein's mRNA [30]. In our experiments with GFP expression in tobacco, possibly the exon junction complex did not occur in all of the GFP RNAs resulting in incomplete splicing and less translation than with the GUBQ1 5′UTR-intron. Future research on the role of the exon junction complex formation in plant cells will be useful to elucidating the mechanism of enhanced gene expression associated with intron splicing.
Levels of GUS expression were comparable in leaves of Gladiolus and Arabidopsis under the G1-1 and CaMV 35 S promoters. Removal of the 5′UTR-intron decreased expression in leaves of both species to background levels demonstrating the contribution of the 5′UTR-intron to expression. Translation efficiency was increased in both species with the presence of the 5′UTR-intron.
Plasmid construction
Isolation of the polyubiquitin promoter from GladiolusGUBQ1, (GenBank accession DQ445914) using the Genome Walker Kit (Clontech) was previously described [15]. The 1.9 kb GUBQ1 promoter (G1-1) and a 0.68 kb intronless version of this promoter (G1-3) were previously subcloned into pUC-GUS and used for transient GUS expression in callus [15]. For Arabidopsis transformations, the GUBQ1 promoter and its intronless version were subcloned into the binary vector pCAMBIA1391Z. For the transient expression assay of tobacco, either G1-1(A) or G1-3 were subcloned into the binary vector pBI121-GFP which contained the gfp gene instead of the uidA gene (Figure 1). G1-1 contains 1898 bp which consists of 600 bp promoter (Gubi1-P), 80 bp 5′UTR-exon (5′U-E), and 1218 bp 5′UTR-intron (5′U-I). In G1-1 (A), the 1218 bp 5′U-I of GUBQ1 has been substituted with the Arabidopsis actin7 gene 5′UTR-intron (541 bp).
The 5′UTR was identified using the 5′RACE & 3′RACE kit (Clontech). Total RNA was isolated from Gladiolus and 2 μg used for RT PCR according to the manufacturer's directions. AP1, an adaptor-specific primer present in the kit, and a ubiquitin gene-specific primer (RT: G1-1 and AS: AGG AAT CCC CTC CTT GTC CTG G) were used for the first PCR reaction of 5′ RACE. The second PCR reaction used AP2, an adaptor-specific primer in the kit, and a ubiquitin gene-specific primer (RT:G1-2 and AS: ATC GAT TGT GTC GGA GCT CTC C). PCR products were cloned into a pTOPO-TA vector (Invitrogen) for sequencing. The 5′UTR sequence was revealed by sequencing and then verified by showing that this sequence was spliced out.
Gladiolus transformation
Suspension cells of Gladiolus cv. Jenny Lee were transformed using the PDS-1000/He gene gun (Bio-Rad, Hercules, CA, http://www.bio-rad.com webcite) [24]. Callus was initiated from plants growing in vitro on Murashige and Skoog's medium (MS) [31] containing 3% sucrose, 1.0 mg/l glycine, 100 mg/l m-inositol, 1.0 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 0.5 mg/l (2.2 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2% Phytagel (Sigma-Aldrich, http://www.sigmaaldrich.com webcite). The callus was used to start suspension cells that were grown in the same medium lacking Phytagel. Both callus and suspension cells were grown in the dark at 25°C. Suspension cells were grown on a gyratory shaker at 120 rpm and transferred every 2 weeks by placing half of the cells into 30 ml of fresh medium.
Suspension cells were co-bombarded at 1200 psi (8.3 MPa) with p35SAc that contains the selectable marker gene phosphinothricin acetyltransferase (PAT) under control of the CaMV 35 S promoter (received from AgrEvo, Somerville, NY) and one of the promoter plasmid DNAs (CaMV 35 S, G1-1, or G1-3). One week after bombardment, cells were placed on MS medium as described for callus initiation supplemented with 3 mg/l bialaphos (Meiji Co., http://www.meiji.co.jp webcite) for selection. Callus was transferred monthly to fresh selection medium, and after about 4 months, surviving callus pieces were transferred to regeneration medium. Regeneration medium is the same as callus initiation medium except with 2 mg/l (9.3 μM) kinetin rather than 2,4-D, and supplemented with 1 mg/l bialaphos. Plantlets recovered after growth on regeneration medium were grown on MS medium without hormones and without bialaphos. The cultures were grown under a 12-h photoperiod using cool-white fluorescent lights (40–60 μmol·m2·s1).
Arabidopsis transformation
Transformation of Arabidopsis thaliana ecotype Columbia plants grown 5 weeks in a growth chamber with a 16 h photoperiod at 22°C were transformed with Agrobacterium tumefaciens strain C58C1 using vacuum infiltration [32]. Hygromycin-resistant T1 plants were selected by planting seeds on MS medium supplemented with 30 mg/l hygromycin. Hygromycin resistant seedlings were then transferred to soil and transgenic plants were verified to be transformed by PCR. The gus gene was amplified using the primer sequences 5′-GTT GGG CAG GCC AGC GTA TCG TG-3′ and 5′-TAA CCT TCA CCC GGT TGC CAG AGG-3′ and thermal cycler conditions of: 1 cycle of 95°C for 5 min and 35 cycles of 94°C for 30 s, 62°C for 30 s, 72°C for 1 min, followed by 3 min at 72°C.
Agrobacterium tumefaciens strain GV3101 was used for transient expression of tobacco (Nicotiana benthamiana). Agrobacterium was cultured for 16 h in liquid medium. Cells were then centrifuged and resuspended in 10 mM MgCl2 at an OD600 of 0.9. Resuspended Agrobacterium cells were vacuum infiltrated into tobacco leaves that had been collected from tobacco plants grown in soil at 22 ~ 24°C for 4-5 weeks under a 16 h light photoperiod. Tobacco leaves were harvested 3 days after vacuum infiltration and used for gfp expression analysis.
RNA isolation and RT-PCR
RNA was isolated from plant tissues using the Qiagen RNeasy kit (Qiagen Inc, Valencia, CA). The isolated RNA was treated with RNase-free DNase (Roche) to remove DNA. Reverse transcription was performed using 1 μg total RNA and 20 μl reaction mix of the iNtRON Power cDNA synthesis Kit (Intronbiotech, http://www.intronbio.com webcite) and AMV reverse transcriptase (Intronbiotech) according to the manufacturer's directions. For identification of 5′UTR-intron splicing in transgenic plants, RT-PCR was performed with the 5′UTR exon specific primer, 5′U-E F (5′-AGG GTT TTC TCA TCC CCA AAT T-3′) and either a uidA or gfp gene specific primer, uidA 200 R (5′- CCT GAT GCT CCA TCA CTT CCT G-3′)/gfp R (5′-AGA AGA TGG TGC GCT CCT GG-3′). For the RT-PCR, 2 μl of each primer (0.2 mM final concentration), and 2 μl of the cDNA preparation were used in the reaction mix (25 μl final volume). The thermal cycler conditions of: 1 cycle of 95°C for 5 min and 30 cycles of 94°C for 20 s, 55°C for 20 sec, 72°C for 1 min. RT-PCR products were cloned into a pTOPO-TA vector (Invitrogen) for sequencing. Quantitative PCR amplification was performed with gene-specific primers and the SYBR Green Master Mix kit (Qiagen) using a Rotor-Gene 6000 real time amplification operator (Corbett Research, Mortlake, Australia). The reaction mix (25 μl final volume) consisted of 12.5 μl of the SYBR Green PCR Master Mix, 2 μl of each primer (0.2 mM final concentration), and 2 μl of the cDNA preparation. The thermal cycler conditions of: 1 cycle of 95°C for 15 min and 45 cycles of 94°C for 20 s, 55°C for 20 sec, 72°C for 30s.
In real-time PCR, the actin gene specific to each plants species being analyzed was used as the reference gene and to determine the possibility of DNA contamination. The actin primer sequences included the following: 1) for the Gladiolus actin gene 5′-CTG CCA TGT ATG TTG CAA TCC A-3′ and 5′-GGA AGA GGA CTT CAG GGC ACC TG-3′ (384 bp), 2) for the Arabidopsis actin gene 5′-GTC CCT GCC ATG TAT GTT GCC-3′, and 5′-GTG GTG AAC ATG TAA CCT CTC-3′ (200 bp), and 3) for the Nicotiana actin gene 5′-TTG GAA TGG AAG CTG CTG GA-3′, and 5′-TCA GGA GGC GCC ACC ACC TT-3′ (202 bp).
All Samples were run three times in triplicate.
Arabidopsis Actin7 gene 5'-UTR intron isolation
The 5′-UTR intron of actin7 gene (Genebank ID, U27811) was isolated from genomic DNA of Arabidopsis thaliana ecotype Columbia. PCR amplification of the 5′-UTR intron was accomplished using the primer sequences 5′-AGG TGA GTC TCT AGA TCC G-3′ and 5′-CCT AAA AAA AAA GTA AAA TGA AAC-3′ and thermal cycler conditions of: 1 cycle of 95°C for 5 min and 35 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, followed by 3 min at 72°C. PCR products were cloned and sequenced.
GUS expression
The specific activity of GUS expression was determined by fluorometric determination of methylumbelliferone (4-MU) [33]. The extraction buffer consisted of 100 mM KH2PO4, pH 7.0 and 1 mM dithiothreitol. Extracts were centrifuged at 16,000 × g at 4°C for 12 min, and a 50 μl aliquot was added to 0.5 ml of assay buffer (1 mM methylumbelliferyl-β-D-glucuronide) for incubation at 37°C. A 100 μl aliquot of the assay buffer containing cell extract was added to 0.9 ml of 0.2 M sodium carbonate after incubation for different lengths of time. Fluorescence of 4-MU was measured using a Bio-Rad VersaFluor Fluorimeter set at 360/40 nm excitation and 460/10 nm emission. The amount of protein in the cell extract was determined using the bicinchoninic protein assay reagent (Pierce, http://www.piercenet.com webcite).
Transgenic plants were assayed for expression of the gus gene that codes for β-glucuronidase (GUS) following the histochemical staining procedure described by Jefferson et al. [33] with some modifications. Shoots were incubated 16 h at 37°C in staining solution [100 mM sodium phosphate, pH 7, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM Na2EDTA, 0.5% (v/v) Triton X-100] with 0.5 mg/l 5-bromo-4-chloro-3-indoly-β-D-glucuronide. After 16 h of staining, the shoots were cleared and fixed in a solution of 75% (v/v) ethanol:1% (v/v) acetic acid.
Three independently transformed plant lines of Gladiolus growing in vitro or three lines of Arabidopsis were analyzed for each DNA construct, and three plants were analyzed for each transgenic line. Three plants of each plant line were used for the mean determination of GUS expression and standard error calculated.
For real time PCR the relative amount of target RNA for each sample was calculated by the statistical analysis method [34].
Southern hybridization
Genomic DNA for Southern hybridizations was isolated from plants using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was digested with Hind III, electrophoresed on a 0.8% agarose gel in 0.5× TBE buffer, and then transferred to a nitrocellulose membrane. For hybridization a 492 bp PCR-amplified gusA gene product was labeled using the PCR DIG Probe Synthesis kit (Roche, http://www.roche.com webcite) and used as the probe. Hybridization and detection of the DIG labeled nucleic acid was performed using the DIG Easy Hyb and DIG Nucleic Acid Detection kits (Roche).
Western blot
Plant leaves were ground in Bradley buffer (50 mM Tris–HCl pH 7.5, 10 mM KCl, 20% glycerol, 0.4 M sucrose, 5 mM MgCl2, 10 mM β–mercaptoethanol, 1 mM PMSF) and centrifuged for 20 min at 13,000 × g, 4°C. The supernatant was used for SDS-PAGE gel electrophoresis, and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with blocking solution (Tris-buffered saline solution pH 7.4, 0.1% Tween 20, 5% nonfat milk). The GFP detection rabbit anti-GFP polyclonal antibody was used as the primary antibody, and anti-rabbit horseradish peroxidase-conjugated was used as the secondary antibody. Immunobiochemical detection was performed using the ECL Plus detection system (Millipore). For quantification of GFP the Multi Gauge Version 3.0 program was used following exposure of the Western blot to Fuji film.
Authors' contributions
KK conceived the idea of studying the effect of the GUBQ1 intron on gene expression in Gladiolus plants, did the Gladiolus expression studies, and wrote the manuscript. YHJ conceived the idea of studying the effect of the GUBQ1 intron on gene expression in Arabidopsis. AY Kim did the real-time PCR and tobacco transient assay analysis. SHP did the GFP analysis. All authors read and approved the final manuscript.
We thank Anne O'Connor for technical assistance with the Gladiolus work. Mention of a trademark, proprietary product or vendor does not imply its approval to the exclusion of other products or vendors that may also be suitable. This work was partially supported by a grant from the Next-Generation BioGreen 21 Program, Rural Development Administration, Republic of Korea (PJ009052) to Y.H. Joung.
Leon P, Planckaert F, Walbot V: Transient gene expression in protoplasts of Phaseolus vulgaris isolated from a cell suspension culture.
Plant Phys 1991, 95:968-972. Publisher Full Text
Norris SR, Meyer SE, Callis J: The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression.
Plant Mol Biol 1993, 21:895-906. PubMed Abstract | Publisher Full Text
Chaubet-Gigot N, Kapros T, Flenet M, Kahn K, Gigot C, Waterborg JH: Tissue-dependent enhancement of transgene expression by introns of replacement histone H3 genes of Arabidopsis.
Plant Mol Biol 2001, 45:17-30. PubMed Abstract | Publisher Full Text
Curi GC, Chan RL, Gonzalez DH: The leader intron of Arabidopsis thaliana genes encoding cytochrome c oxidase subunit 5c promoter high-level expression by increasing transcript abundance and translation efficiency.
J Expt Bot 2005, 56:2563-2571. Publisher Full Text
Mascarenhas D, Mettler IJ, Pierce DA, Lowe HW: Intron-mediated enhancement of heterologous gene expression in maize.
Plant Mol Biol 1990, 15:913-920. PubMed Abstract | Publisher Full Text
Leuhrsen KR, Walbot V: Intron enhancement of gene expression and the splicing efficiency of introns in maize cells.
Mol Gen Genet 1991, 225:81-93. PubMed Abstract
Maas C, Laufs J, Grant S, Korfhage C, Werr W: The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold.
Plant Mol Biol 1991, 16:199-207. PubMed Abstract | Publisher Full Text
Bourdon V, Wickham A, Lonsdale D, Harwood W: Additional introns inserted within the luciferase reporter gene stabilize transgene expression in wheat.
Plant Sci 2004, 167:1143-1149. Publisher Full Text
Callis J, Fromm M, Walbot V: Introns increase gene expression in cultured maize cells.
Genes & Dev 1987, 1:1183-1200. PubMed Abstract | Publisher Full Text
Clancy M, Vasil VL, Hannah C, Vasil IK: Maize shrunken-1 intron and exon regions increase gene expression in maize protoplasts.
Plant Sci 1994, 98:151-161. Publisher Full Text
Wilmink A, van de Ven BCE, Dons JJM: Activity of constitutive promoters in various species from the Liliaceae.
Plant Mol Biol 1995, 28:949-955. PubMed Abstract | Publisher Full Text
Vain P, Finer KR, Engler DE, Pratt RC, Finer JJ: Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.).
Plant Cell Rep 1996, 15:489-494. Publisher Full Text
Vasil V, Clancy M, Ferl RJ, Vasil IK, Hannah LC: Increased gene expression by the first intron of maize shrunken-1 locus in grass species.
Dugdale B, Becker DK, Harding RM, Dale JL: Intron-mediated enhancement of the banana bunchy top virus DNA-6 promoter in banana (Musa spp.) embryogenic cells and plants.
Plant Cell Rep 2001, 20:220-226. Publisher Full Text
Joung YH, Kamo K: Expression of a polyubiquitin promoter isolated from Gladiolus.
Plant Cell Rep 2006, 25:1081-1088. PubMed Abstract | Publisher Full Text
Fu H, Kim SY, Park WD: High-level tuber expression and sucrose inducibility of a potato sus4 sucrose synthase gene require 5′ and 3′ flanking sequences and the leader intron.
Plant Cell 1995, 7:1387-1394. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lu J, Sivamani E, Azhakanandam K, Samadder P, Li X, Qu R: Gene expression enhancement mediated by the 5′UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants.
Wei H, Wang ML, Moore PH, Albert HH: Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants.
J Plant Phys 2003, 160:1241-1251. Publisher Full Text
Goodall GJ, Filipowicz W: Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.
EMBO J 1991, 10:2635-2644. PubMed Abstract | PubMed Central Full Text
Rose AB, Elfersi T, Parra G, Korf I: Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression.
Plant Cell 2008, 20:543-551. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
McElroy D, Zhang W, Cao J, Wu R: Isolation of an efficient actin promoter for use in rice transformation.
Plant Cell 1990, 2:163-171. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Wang J, Oard JH: Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems.
Plant Cell Rep 2003, 22:129-143. PubMed Abstract | Publisher Full Text
Rathus C, Bower R, Birch RG: Effects of promoter, intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts.
Plant Mol Biol 1993, 23:613-618. PubMed Abstract | Publisher Full Text
Kamo K, Blowers A, Smith F, Van Eck J, Lawson R: Stable transformation of Gladiolus using suspension cells and callus.
Rose AB, Beliakoff JA: Intron-mediated enhancement of gene expression independent of unique intron sequences and splicing.
Plant Phys 2000, 122:535-542. Publisher Full Text
Rose AB: The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis.
Plant J 2004, 40:744-751. PubMed Abstract | Publisher Full Text
Rose AB, Emani S, Bradnam K, Korf I: Evidence for a DNA-based mechanism of intron-mediated enhancement.
Samadder P, Sivamani E, Lu J, Li X, Qu R: Transcriptional and post-transcriptional enhancement of gene expression by the 5′UTR intron of rice rubi3 gene in transgenic rice cells.
Wiegand HL, Lu S, Cullen BR: Exon junction complexes mediate the enhancing effect of splicing on mRNA expression.
Proc Natl Acad Sci USA 2003, 100:11327-11332. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Nott A, Hir HL, Moore MJ: Splicing enhances translation in mammalian cells: an additional function of the exon junction complex.
Genes & Dev 2004, 18:210-222. PubMed Abstract | Publisher Full Text
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures.
Physiol Plant 1962, 15:473-497. Publisher Full Text
Bechtold N, Pelletier G: In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration.
Methods Mol Biol 1998, 82:259-266. PubMed Abstract
Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
EMBO J 1987, 6:3901-3907. PubMed Abstract | PubMed Central Full Text
Pfaffl MW, Horgan GW, Dempfle L: Relative Expression Software Tool (REST©) for groupwise comparison and statistical analysis of relative expression results in real-time PCR.
Nucleic Acids Res 2002, 30:E36. PubMed Abstract | Publisher Full Text | PubMed Central Full Text