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This article was originally published in MDDI on August 2008.
The green writing is on the wall: it is time for medical manufacturers to consider the sustainability of their products, packaging, and production processes. In the consumer world, Wal-Mart is undergoing a major effort to adopt sustainability and even hired the ex-head of the Sierra Club as a consultant on this topic. Clorox recently announced its Green Works line of greener household cleaning products. Companies with a strong presence in the medical field like Kimberly-Clark and Philips have long been addressing the problem.
This author's interviews with medical product consumers around the country have revealed concerns about sustainability. Consumers consistently bring up the topics of global warming or green in discussions about new products that, as recently as two years ago, would have been solely focused on efficacy and usability.
Even if you are not doing something about getting greener, your customers are. Alegent Health, a company with nine hospitals and 8600 employees, has recently named a vice president of sustainability. When faced with a choice of medical products of similar cost and efficacy, it is likely that customers will purchase the greener product, especially if manufacturers have added green to their brand attributes in a way that customers see has real meaning.
Sustainability broadly means considering the environmental effect of a product throughout its life cycle, not just in its creation and initial use. And it is a daunting topic for the uninitiated. Although RoHS and other legislation in Europe have brought some sustainability issues to the forefront, it is understandable that many medical manufacturers have been reluctant to embrace sustainability. The device industry is notoriously slow to make changes. In addition, the industry is sometimes exempted from the legislative restrictions required in the consumer marketplace and is therefore less likely to pursue such change. Further, sustainability has an image of increasing costs.
The good news is that there is a lot of low-hanging sustainability fruit that can be harvested by applying common sense principles and a sustainability-conscious eye to the product life cycle.
This article presents practical advice to designers and manufacturing engineers about how to improve the sustainability of device products. Be prepared to be surprised, as have many in the consumer world—you might just find out that it makes good business sense too.
Start Here: Map the Product Life Cycle
Understanding how manufacturers can use sustainability requires mapping a product's life cycle. This includes raw material extraction; all processing and manufacturing; actual use; and disposal, reuse, or recycling.
Creating a sustainable product is an attempt to reduce the environmental footprint at each stage with some kind of change. Changes do not have to be massive to have a positive effect. For instance, Philips Healthcare considers a product a Green Flagship if it achieves 10% improvement over its predecessor or competitor. A review of Philips products with such status indicates that most achieved improvements in the 25–35% range.
OEMs usually focus on improving efficacy and usability, and minimizing trauma and cost. Like these factors, sustainability has the biggest influence at a product's conception. Many sustainable qualities of a product are baked in during the innovation and design stage.
For medical products, the business model is also important. For example, a one-time-use disposable consumes more resources than a reusable product. Of course there may be clinical, product, or sterility issues that require disposability. Because of such factors, the approaches to sustainability from the general consumer or industrial world do not always translate well to medical products.
Quantifying Design Alternatives
Once a specific device's life cycle is understood, an OEM can begin identifying the places to lower its environmental impact. The team should quantify the effects of various choices.
One way of enumerating a particular design's effect is to use software such as Life Cycle Assessment (LCA). This software draws on carefully researched databases, allowing manufacturers to estimate the effect of one type of plastic over another, the weight and material type of packaging, or shipping options. Leaders in LCA software are European companies with products such as SimaPro and GaBi.
Although large companies may already own such software or think nothing of purchasing it and training people on how to use it, the software may not be the right place for most device manufacturers to start.
Hans van der Wel, Philips Healthcare's manager of ecod esign and sustainability, helps run its Green Flagship program. He says the best method for starting out is to "keep it simple. Start with a spreadsheet based on simple indicators. We call ours green focal areas [and] include qualities like the amount of materials, energy, and hazardous substances used." He says it took Philips 10 years to get to its Green Flagships program.
Example: An RF Surgical Tool
An example product demonstrates how device designers might approach sustainability. A product system includes a disposable and an energy-supplying console. The following section explores the effects of changing these system elements, which are typical to many medical products.
In evaluating the effect of the various components that make up the whole product, designers need to use real impact data. The LCA software makes finding data easy. It is probably the best long-term tool, but it requires a commitment of money and training that might slow initial efforts. Alternatively, the Okala Guide is inexpensive ($12) and has a useful table of impact factors that covers common materials and processes. It is used in this example. The Okala guide combined with a simple spreadsheet may be good tools to get started.
To make this example easy to understand and illustrative of the kinds of improvements possible, not every material or process is compared. In some places, system elements are combined and given overall numbers to ease the reading of the tables.
The analysis focuses on areas in which manufacturers can have the most influence. In practice, when you consider the whole product, the actual impact reduction achieved might be lower than the numbers shown here. However, on an established product, an overall 20–30% reduction is relatively easy to achieve.
Product Description.
A hypothetical radio-frequency (RF) tool for clamping and cauterizing surgical wounds is being considered for redesign. The company hopes to improve sustainability. The product is a system consisting of a disposable handpiece and an RF energy generator and controller.
The Disposable Handpiece: Consists of a plastic-and-metal handle with integrated mechanisms that provide mechanical advantage to the surgeon's grip during the procedure, and a wiring harness to connect to the console. The handle is currently single-use (all parts) and is contained in sterile packaging. It is manufactured at one location but used in all major global markets.
RF Energy Generator and Controller: Consists of a piece of capital equipment that is a power and control source for the disposable. It is based on five-year old electronics and display technology, has no field-upgradable features, and is intended to last five years. The console is built into its own hospital cart.
Two levels of improvement are considered: first, a few options f or redesign of the disposable, and second, a redesign of the reusable energy-generating console. To begin, readers should understand the existing product's ecological footprint to accurately compare new design approaches.
Calculating the Footprint.
Everything that is used to make, use, or dispose of a product is scored based on its environmental effect. This is calculated using a rating called impact factor. Impact factor numbers have been gathered or researched and reduced to a standardized unit by an agency such as the makers of LCA software or compiled in resources such as the Okala guide used here. This factor is based on how the particular parameter is used in the product (e.g., per lb material, kWh of energy, tn/miles of transportation, etc.). Totaling the scores yields an overall product rating. In this example, units are in Okala milli points. Impact factor units must be the same for all contributors. Values from sources that do not share units cannot be mingled.
The RF device example looks at the effect for the entire life of the product—about five years. During this time a typical user buys one console and uses 10 disposables per week, yielding a use of 2600 disposables per console lifetime. This means that of the roughly 212,000 impact points, the 2600 disposables used over the product life contribute about 196,000 points. It is important for designers to consider the overall system usage, not just individual parts, in evaluating and comparing the various redesign choices. With this calculation in mind, consider the following possible changes to the hypothetical product.
Scenario 1
Make the Disposable Part Weigh Less (console unaffected). This scenario simply considers using improved design optimization tools such as finite-element analysis (FEA) to reduce the material needed for both the plastic and metal components (without compromising function).
A slight weight reduction has various virtuous effects. Less material is used, which reduces environmental impact. In addition, lower material cost helps offset the increased design and validation costs of a lighter handpiece. Packaging can also get a little lighter to reduce shipping cost and impact.
A modest change to the product, requiring no major changes to the way it is made or used, yields a small but meaningful 6% reduction in impact over the product's life.
Scenario 2
Make the Disposable Weigh Less and Enable the Wiring Harness to Be Reusable 20 Times. The development team notices that almost half of the disposable's impact comes from the copper wiring in the leads that connect it to the console. The leads are redesigned to be sterilized and used 20 times instead of just once. The copper content remains the same, but the insulation needs to be beefed up to make the parts rugged enough to withstand reuse. Also, now they will not be packed with every disposable but instead be shipped one set per box of 20 hand pieces. Overall, the product and packaging are lighter, further reducing impact score. A factor must be added for the sterilization by the hospital.
Reusing the wiring harness has a very significant effect. Now the impact is reduced 44% overall from the original product. This redesign does, however, require a change in how the product is sold (box of 20 handpieces with just one wiring harness enclosed) and in how it fits into the hospital's overall workflow. Hospitals must sterilize, manage, and inventory a wiring harness for 20 uses. But copper is also expensive, so now the cost to provide the function of 20 handpieces has decreased. Such savings could be passed on to users in exchange for the task of sterilization, without negatively affecting the manufacturer's profit per handpiece. It is a balancing act, because the cost of sterilizing at the hospital may outweigh any savings.
Although the market may not yet be ready to make such a change in how it handles wiring harnesses, the example shows how such a change contributes to making the overall system green. As some large hospital groups become serious about being more sustainable, they may be prepared to make these kinds of changes in the near future.
Scenario 3—Build on Scenario 2 by Redesigning Electronics and Choosing Lighter Materials for the Enclosure.
Now the development team turns its attention to the console and notices that the energy used while the machine is on (usually for a four-hour procedure) affects every disposable. Modern electronics are not only RoHS compliant, but also offer a more efficient package in terms of PCB size. Furthermore, new electronics consume half the energy of older electronics (due to improved sleep modes between uses during the procedure). Now that the console is smaller, it can be built into a lighter, portable enclosure, rather than integrated into a heavy cart. Such changes have a significant effect on both disposable and capital system elements, and yield an overall 52% reduction. Notice that if further changes are made to the console electronics to reduce copper wiring by 20–50% (perhaps by some novel pulsing technique or a change in the frequency of the RF), it would further improve the product's impact score.
A simple spreadsheet scenario like the tables allows a team to see possibilities for redesign. This is obviously a highly generalized example. Only your technical and marketing teams know where the opportunities lie for your specific product in its clinical efficacy and marketing acceptability.
The Disposable Matters More
In this example, it is assumed that 10 disposables are used per week per console—not an unreasonable assumption for this kind of surgical tool. As noted, this means that over a five-year design life, 2600 disposables are used. Therefore, even a small improvement in the disposable has a magnified effect. By contrast, if the design team halves the impact of the console it hardly reduces the system's overall impact. Designers must consider the effect of the whole system's use.
Although the case study shows that focusing the redesign effort on the disposable has the most affect on environment, there are still things the manufacturer can do to the reusable portion of the product that can further reduce its impact. In the example, 50% more efficient electronics in the RF energy generator lowered the energy portion of the product's impact 2600 times because each use of the disposable cost 50% less energy.
This exercise shows how changes such as lowering energy use through a better sleep mode for the console can have a greener consequence than, say, using a lighter plastic on the enclosure. It's not always the obvious changes that have the most benefit, and unfortunately, finding the changes that have the greatest potential are not formulaic. Each product will have very different aspects that must be rethought.
Perhaps as an industry we need to reconsider what it means to be green. As Wendy Jedlicka, a sustainable packaging expert, puts it, "The idea that you have to wholly embrace eco like a religion is shortsighted and frankly not sustainable. We need to get everybody doing a little bit of something to mitigate what we are doing right now; then we can keep improving."Reference
1. Philips Medical Green Flagships [online] (Amsterdam [cited 28 May, 2008]); available from Internet: www.medical.philips.com/main/company/sustainability/ green_flagships.


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"Some apartaments in a separated building, where we stayed, are overlooking moutains and a small practicing..."

subset: commoncrawl


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|Forum topic by Howie
|posted 01-28-2011 02:28 AM
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Built mommie a computer desk from plans out of a WW magazine. UNFORTUNATELY, I made the mistake of buying some chinese plywood from HD. I sanded it(100,120,150 and no, I did not go thru the veneer)Minwax prestain then I stained it(minwax) let dry for two days…minwax clear poly top coat….....now the problem…several sections of the plywood is not dry after 24 hours….duh! It feels like glue is leaking out of the PW(stickey). Another thing…it had white spots show up like there was grease or oil on it. I'm positive I did not get anything on it. Makes me wonder if this plywood came from the same factory the drywall came from. Bah Humbug(for lack of any other words I can put on here )
-- Life is good.


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Xerox Designated RSA's WebCRD as 'Optimized for FreeFlow'
Monday, October 27, 2008
Press release from the issuing companyRochester, NY, October 26, 2008 - Rochester Software Associates, Inc. (RSA) announced its WebCRD web-to-print solution has been designated as "Optimized for FreeFlow" by Xerox Corporation. RSA's software has earned this prestigious Xerox Business Partner distinction by tightly integrating its WebCRD solution with Xerox FreeFlow Digital Workflow Collection. Xerox's FreeFlow Digital Workflow Collection operates as the "central point of control" in a digital print shop environment by automating printing and administrative operations.
WebCRD is RSA's award winning web submission and print production workflow software used by hundreds of corporations, universities, government, financial and medical organizations. WebCRD was recognized by InfoTrends and On Demand as the Best of Show in the Web-to-Print category this year for the second consecutive year.
WebCRD leverages the Xerox FreeFlow SDK architecture by:
- Integrating with Xerox FreeFlow Process Manager to automate workflows and improve customer productivity
- Integrating with Xerox FreeFlow Process Manager, to automate printing of RDO files via RSA's WebCRD
- Creating Xerox Printing Instruction Format (XPIF) Job Tickets, for automated print and finishing functionality.
Xerox customers can now maximize their investment in Xerox's FreeFlow and RSA's WebCRD to automate workflows, increase print center productivity and improve customer satisfaction. WebCRD, Optimized for FreeFlow, will be featured in the Xerox booth 1219 at Graph Expo 2008, October 26-29 in Chicago, IL.
Industry News
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I just looked up and noticed its 2013 which means that a vast majority of ®eformers have earned tenure as ®eformers while achieving various levels of effectiveness. Think of your favourite ®eformer, what their goal was when they started, did they reach their goal? Is your favourite ®eformer still fighting for their cause after 2 or 3 years of proving they were ineffective at creating the change they said they would? Did they get fired or get tenure?
Considering the disproportionate funding in favour of ®eformers vs the others, they cannot cite a lack of funds. Has your favourite ®eformer backed down a little bit in their rhetoric and tried to temper expectations like a corporation before a bad earnings report? Have you ever heard of a ®eformer being fired for poor performance? notyetLAUSD proposes a 4 year tenure track for ®eformers working inside and outside LAUSD. If you can't make the changes you want, move over and let someone else try. Fired!
In other news:
Green Dot charter just announced they are going to reconstitute themselves at Locke High School in an effort to show the use of self-immolation and, an undying faith in reincarnation of the self, is needed to create meaningful change at high need public schools.
A lame donkey compromise:
Can we just get over tenure talks and LIFO, move tenure to 4-6 years with no more than two school transfers and 3 consecutive years at the site where tenure is given? Can we all agree that it sucks when a great young teacher is laid off and a crappy old one is kept, but over the past 4 years of laying off less experienced teachers and concentrating its number of experienced teachers, LAUSD was able to boost API at a far faster rate than other districts. (I know most of the boost over the past few years has been due to increases in the use of the CMA, but I'd like to live in a fantasy world where having more students in classes with long-term committed teachers might have had an effect too). I'm not opposed to the district going back using incentives at hard to staff schools to prevent the need for a Reed settlement.


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patterns > Knotions Magazine > Knotions, Fall 2016
> High Tide Socks
High Tide Socks
I've always been fascinated by high tides, when the beach slowly disappears and the sea reaches the city before withdrawing again and you have to walk kilometers on the beach to find it. Tides have always punctuated my vacations so I wanted to design those socks to knit at the beach while watching the movements of the sea.
These socks are knitted in the round on four double pointed needles, but the experienced knitter can easily adapt the pattern for one or two circular needles. They are knitted top down, with a round heel and a wedged toe. The stitch pattern is made of stockinette stitch and elegant wavy cables over garter stitch.
Foot circumference 7.5 (8.5, 9.5)" or 18 (20.5, 23) cm
Shown in size 8.5"
The leg and foot length are both adjustable
Knit Picks Stroll Fingering (75% Superwash Merino Wool, 25% Nylon; 231 yards 211 meters/50 grams): 25028 Wonderland Heather, 2 balls
Size US 1 (2.25 mm): 4 dpns
Cable needle; tapestry needle
30 sts and 38 rows = 4" 10 cm in St st after wet blocking.
49428 projects
stashed 39939 times
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Welcome Gentles All. Your horse has brought you to No. 1623 Shakespeare Place. The Place Where There's a Space for Anything Shakespeare. Any & All Comments Welcome
Monday, September 20, 2010
Book Review: Will Power by A. J. Hartley
Will Power, second in a series of fast-paced fantasy novels by A J
Hartley, makes its debut on the bookstore shelves today. I write "series" in the
hopeful assumption that Hartley has plans for continuing the saga of one Will
Hawthorne--actor, poet, playwright, con man, and now full-time reluctant adventurer. Hawthorne's somewhat checkered rise to stardom as a young Elizabethan-style actor of plays and self-proclaimed pithy purveyor of pentameter poesy, was rudely interrupted in the first installment, Act of Will, leading to new employment as front man for a band of, in Will's modest opinion, much too heroic adventurers.
Stylistically, Professor Hartley has created a somewhat eclectically archaic world
for us to adventure through in accompaniment to his heroes. An entertaining mix of places and individuals with the flavor of the Elizabethan, the Gothic, and the sometimes hard to categorize populates the narrative, written in the first person from Will Hawthorne's perspective. But our inability to 'peg' this time and these places, while at the same time being more than faintly all too familiar with them is the result of a clever device on Hartley's part. And it's the use of that very device that makes it an unusually fresh approach in my opinion. Hartley is able to remain true to his atmosphere with rich and vivid descriptions of the surroundings, situations, battles, creatures, and ancient weaponry, yet allows the reader to settle into an easy, comfortable, narrative realm, through the modern linguistic expressiveness and colloquialisms of his lead character, Will--and far from clashing, the style winds up complementing itself over and over.
Following the events through the eyes of Will Hawthorne, Will Power transports us to ancient lands of Goblins who use bears as horses, wolves that seem to understand what you're thinking, and a gleaming White City where King and court seem all too concerned with outward appearances. Therein lies a theme commented on by Hartley. But the commentary isn't heavy handed. It's intertwined so well within the story line that the denouement, held close enough to the vest to more than support the lessons we might learn, still comes as a shock to the senses, surrounded by the events Hartley so deftly and vividly describes for us. In reality, though we might sometimes wish to ignore their existence, we Know these strange characters for who and what they are--and the realization can come to us as a little unsettling, even though we must nod in assignation as we read. This is one of the qualities I have come to admire about Hartley as a writer; he instructs as well as entertains.
Hartley has also seen fit to aptly furnish his saga with a back story. In the author's words:
Like the first volume, Act of Will, it [Will Power] has been translated from
the original Thrusian--as preserved in the now famous Fossington House
Papers--with the aid of notes left by the Elizabethan translator Sir Thomas
Henby. As readers of the first manuscript will quickly see, the second volume is
different in key respects from the first, and raises still more vexing questions
of provenance, locale, and issues of how much of the narrative--if any--is
derived from fact.
He then goes on to promise the results of further investigation in a series of
academic papers to be published in issues of Philological Quarterly
--HA!--Though he doubts that a general reader would be very much interested.
-- What a hoot.
This is the type of well-rounded attention to detail I came to expect from Hartley, becoming familiar with his work after having fortunately stumbled upon his mystery novel, What Time Devours. See my review. And it's what makes Will Power read with the veracity of a mysterious and exciting in-but-out-of-this-world historical chronicle, rather than pure fantasy.
Although I must admit, upon finishing Will Power, I immediately began a quest
to find book #1 --Act of Will , Will Power certainly holds up to
its billing as a 'stand-alone' fantasy novel. But trust me, if you're a fantasy
fan, you too will be looking for more from Professor Hartley, past or future. This book, as well as all of his others, no matter the genre, is recommended reading. JM
From Tor/Forge Publishing: British-born writer A. J. Hartley is the Distinguished Professor of Shakespeare in the Dept. of Theatre and Dance at the University of North Carolina at Charlotte. As well as being a novelist and academic, he is a screenwriter, theatre director, and dramaturge. He is married with a son, and lives in Charlotte.
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Category Archives: MVC4
Single Sign-On (SSO) implementation in ASP.NET MVC
To start with any secured web application, the developer needs to work on the implementation of the authentication functionality. If any user needs to enter into multiple secured web application on same domain in .NET framework, he needs to login … Continue reading
Performance Optimization – Bundling and Minification in ASP.NET MVC 4
Before getting you into the details of performance optimization, let me give a brief note on Bundling and Minification of JS/CSS files. What is a Bundle? Bundle is simply logical group of files that could be referenced by unique name … Continue reading


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ETSY! ::: Where I sell my handcrafted goodies. Mostly jewelry & photography, but occasionally other cool bits too.
deviantART ::: Older art & photography. Click here to look at a spectacular Fractal Calendar I designed & that's for sale. (It's the only item I have for sale on DA).
ArtWanted ::: Selected photography by me that you can comment on.
Flickr ::: A TON of photos & graphic art. Many many photos of my cats, plus random creative bits, WIST goodness, and more. You can comment on everything here!
indiepublic ::: Profile & blog.
myspace ::: Profile & blog.


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Views: 549, Replies: 5 » Jump to the end
Jan 6, 2011 3:42 PM CST
|I was looking at my lilies and was wondering about the hole in the cut stalk. Has anyone had a problem in the past with water going thru the hole in the stalk to the center of the lily bulb and rotting the bulb center? I have had a few bulbs that did not come up after the first year and when I dug ( two years later) the bulb up it was gone.
Jan 6, 2011 9:36 PM CST
|Good question... Some people think this is a problem but I'm really not sure that it is. I cut my stalks off at about six inches and pull them out in the spring when new growth appears. If your soil is poor draining and you think water is being held in the stalk, you might try pinching foil caps over the cut stalks in the fall. That should keep water out.
Jan 6, 2011 10:01 PM CST
|Personally I think it's a valid concern. That's why I quit cutting my stalks down on my lilies in the fall. I just leave my stalks on over the winter and then gently tug them off in the spring, or cut them if they don't come out easily.
My business:
Click here for Siberian Iris Gardens!
Jan 6, 2011 10:10 PM CST
|That is ok if you don't have problems with botrytis. It seems prevalent in our maritime climate so I try to get rid of as much stalk as reasonable and still be able to see the stub!
Jan 6, 2011 10:15 PM CST
|None here, so far, and we don't have the dreaded lily beetle. I have no idea why, because it's to the west of us in Rochester NY, and direct east in Mass. My lilies are in rows between the irises, and the irises get sprayed a lot. Maybe that's why?
Because I must overhead water, I spray the lilies and lilacs with baking soda. Could that be preventing it? I'm really not up on botrytis.
My business:
Click here for Siberian Iris Gardens!
Jan 6, 2011 10:32 PM CST
|Yes to all... fungicides including baking soda will prevent it or at least keep it to a minimum. Around here it seems to always be raining when I need to spray. Throw in a hail storm to damage the leaves and an infection is a sure thing.
On the other hand some lilies are really resistant to it. For example, Scheherazade.
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We've been traveling for several months now.
I keep thinking I should just start a blog about our travels, and maybe I will, but I keep having experiences that I want to share. So, I think I'll start sharing them anyway, and if I decide to migrate some posts to another blog later, that's fine.
And in the meantime, if anybody doesn't want to read about travel topics, feel free to skip topics that are not interesting to you.
We started our trip in Panama. Then we went to Uruguay. We felt really comfortable in Uruguay, randomly, so we decided we want to stay longer. We went to Argentina for a little vacation, and now we're back in Uruguay for the next 5 months; we're still planning to visit other places, but we'll have our house here as a home base.
So, that's what we're doing.
Saturday, October 26, 2013
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Meet Handle. This is Boston Dynamics' brand-new robot and if we're being completely honest it's probably their scariest yet for a few very simple reasons.
Firstly the wheels. What was wrong with feet, eh? Not content with our perfectly usable human feet Boston Dynamics have given Handle two wheels instead and you know what, it turns out they're just about better than feet in every single way.
Handle's not only able to go faster than us over almost any terrain but it's also able to turn quicker and even jump.
That's right, this robot can jump, and jump really well.
The research robot stands a towering 6.5ft tall and can travel at 9mph while jumping 4 feet in the air.
It's able to roll down stairs, cope with seriously uneven terrain and even lift up objects that weigh 100lbs.
It also has a range of around 15-miles on a single charge so unless you're planning on doing some marathon training anytime soon Handle will be able to catch-up with you.
That's something to think about isn't it.
Boston Dynamics' Terrifying Robots:
Introducing SpotMini
Introducing WildCat
Petman Tests Camo
Cheetah Robot runs 28.3 mph; a bit faster than Usain Bolt
Atlas, The Next Generation


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This item is a pink divided relish dish in the "Poinsettia" pattern from Jeanette Glass. The dish is 1 3/8" deep, 8 1/4" long, with a body width of 5". The slightly ribbed sides and bottom are decorated with the "poinsettia" pattern and the interior and handles are smooth. There is a 1/2" long abrasion on the bottom of the dish that does not touch the design with no other wear or damage present. Please feel free to contact us with any questions or to make an offer.
21 other shoppers are interested in this item
Antiques, Collectibles


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Beyoncé Gets Temporary Tattoos for Fashion Campaign
Beyoncé has added some serious ink to her body, but it's only temporary. She's been photographed with elaborate tattoos on her back, arms and legs to promote a new collection of temporary tats being marketed by her Dereon fashion line. According to Women's Wear Daily, Beyoncé and her mother, Tina Knowles, are partnering with airbrush makeup firm Temptu for the collection, which will go on sale Nov. 1 at Sephora stores and on Dereon.com. Tina Knowles tells Women's Wear Daily, "With a sixties-pinup-girl meets-futuristic-biker-chick theme, Beyoncé and I really wanted to give our Dereon fall 2010 campaign a tough edginess."
Copyright 2010 ABC News Radio
Monday, September 27, 2010 at 10:34AM by Jeanette Torres Permalink
tagged Beyonce, Dereon, Tattoos, Tina Knowles in Entertainment General


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It is really much difficult to stay in a bloody battlefield. Since everything is in chaos, you would not be able to recognize who your friends and your foes are. You would not really know if the one wearing your shield and armor is really your kind. You would not even believe that the one wearing your mask is your adversary. Even the closest and truest of your kin would be the one to sell your honor to the other side.
Life is our own bloody battlefield. You are your only ally in the ground. You are the only one who you could trust the most. Without knowing yourself, primarily would make you such a great deal of a loser in the combat zone. Without equipping yourself with a strongest of heart and a sharpest of mind would mean entrapping yourself to the blades of your opponent's sword which thirsts for your own flesh and blood like the fangs of the starving lions waiting for their time to feast on.
Warriors in combat showcase all of their skills and agility coupled with their unmatched physique. Like the Spartans, you should stand firm and defend your own honor and dignity. Even if you are already on the losing end, you should never give up to your rival's dominion. Never surrender. Like them, you should fight and defend your honor to your last breath. For if you don't, you will never find your way out of that battlefield where you are in and never see victory! Ever again!


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Mumbai: The makers of forthcoming film `Madras Cafe` initially wanted to cast actress Freida Pinto along side actor-producer John Abraham, but she couldn`t take up the project.
Freida rose to prominence as Latika in the 2008 British Indian drama film `Slumdog Millionaire`. The actress became an overnight sensation after the film won a bevy of international awards.
Since then she has worked in international projects like Woody Allen`s `You Will Meet a Tall Dark Stranger`, `Rise of the Planet of the Apes` opposite James Franco and `Trishna`.
Earlier, apparently director Shoojit Sircar had approached Freida for his upcoming John Abraham starrer political thriller `Madras Cafe`, reportedly set against the backdrop of the Sri Lankan civil war of the 1990s.
"We had initially approached Freida Pinto who had loved the script and expressed her interest in doing the film. She (Freida) had Woolly Allen`s film so she couldn`t do it," John said.
As Freida couldn`t do the film, the makers later approached actress Nargis Fakhri. The `Rockstar` actress is playing the role of an international journalist.
"She (Freida) called me and said it is courageous of me to make a film like `Madras Cafe` because India needs to see more films like that. I took that as a compliment. It is nice to hear that," John said.


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By Rohingya Mirror & Rohingya Eye | December 27, 2016
Maungdaw — A Rohingya man who refused to demolish his own home was shot at head by Myanmar's Border Police (BGP) in northern Maungdaw on December 25, while the BGP continues to demolish more Rohingya homes across the Maungdaw District reportedly on the order by the Rakhine (Arakan) State Administration, it has been reported.
The incident happened at the village of 'Wa Chein' (in northern Maungdaw) at around 11 am on Sunday and the victim is identified as Zahid Hussein s/o Aamir Hussein.
"The victim was fortunate enough to escape death as the bullet hit at the left side of his head but lost his consciousness and fell down immediately. He is now in critical condition being treated in the clinic in 'Taman Thar' village, according to the reliable sources in the region.
"The BGP ordered the villagers of 'Wa Chein' to destroy 30 homes on the pretext that they were not included in the 'Household Map.' The villagers didn't demolish their homes. So, the BGP raided the village on December 25.
"They arrived at Zahid Hussein's home and ordered him to Demolish his own home. He replied that he couldn't do so without looking at the situation and would do so only when others do. So, the BGP forced started and torturing him and another BGP man shot him at his head from 5 yards away.
He fell down and became critical. He is now being treated at Taman Thar Clinic", said a local on the condition of anonymity.
After the incident happened, the BGP forced other six families in the village to demolish their home using threats and terror and then ordered to demolish the remaining.
Some homes demolished at the village of 'Wa Chein' belong to the following people.
1) Ali Hussein s/o Noor Hussein
2) Abu Ahmed s/o Noor Mohammed
3) Zahid Hussein s/o Aamir Hussein
4) Abu Alam s/o Sultan Ahmed
5) Mohammed Shafi s/o Abdu Jalil
The numbers of the people becoming displaced are alarmingly increasing and many people are trying to flee to Bangladesh as the BGP forces continues to demolish more and more Rohingya homes across Maungdaw a2nd Buthidaung Townships reportedly acting on the Order by the Rakhine State Administration (controlled by NLD).
A list of homes and shops destroyed recently at some other villages is as follows.
On December 22, 3 houses were demolished at 'Zuthar Hali.' The house owners are:
1) Mohammed Hussein (31), s/o Abul Hussein
2) Fedaan Ali (25), s/o Ghaffar
3) Noor Mustafa (42), s/o Nurul Hoque
On December 22, 2 shops were demolished at 'Faari.' The owners are:
1) Inayatullah (41), s/o Halimullah
2) Dolu (40), s/o Abu Nu'man
On December 24, 5 houses were demolished at 'Maudi' village. The owners are
1) Zahid Alam s/o Sayed Alam
2) Noor Alam (20), s/o Mohammed Ali
3) Mohammed Sharif (20), s/o Shamim Jalal
4) For Alam (24), s/o Mv Hakim Ali
5) Ayub (25), s/o Sayed Noor
On December 24, six houses were destroyed at 'Fulaya' hamlet of 'Kwan Thi Pin' village. They belong to:
1) Zafar (30), s/o of Rashid Ahmed
2) Mohammed Rashid (25), s/o Sirajullah
3) Dil Mohammed (50), s/o Rashiduddin
4) Faisal (24), s/o Islam
5) Osman (23), s/o Nooru
6) Mohammed Ayub (25), s/o Noor Mohammed
On December 24, two mosques, two shops and 8 homes were demolished by the BGP at the village of Kyar Gaung Taung locally known as Rabailla.
Mostly, the BGP have forced the Rohingya villagers to dismantle their own houses and shops at gun-points or by means of using threats of imprisonment. In the occasions when the locals resisted, the BGP themselves have demolished many homes. It has also been reported that the BGP indulge in plundering Rohingya properties and livestocks during their raids on Rohingya villages to destroy homes.
More than 50,000 Rohingya people have already been displaced by the brutal military offensives in northern Maungdaw, a region which has been sealed off from international media and humanitarians, since October 9, 2016. Adding to that, the government actions to demolish more homes are leaving many people without any shelter in this winter and going by the emerging reports, many more people in Maungdaw are trying to flee to neighboring Bangladesh for the safety of their lives.
"On one hand, they unlawfully killed, torured and arrested hundreds of innocent people and raped many women. On another hand, they burnt down thousands of homes. And then, they removed fences surrounding our homes. And now, they are destroying our homes. More people are trying to flee to Bangladesh. Where are the laws and humanity?" exclaimed an elderly in northern Maungdaw, while speaking to Rohingya Vision TV.
The houses and shops being destroyed in the rural villages are not modern buildings that require officials' permission(s) but traditional country-type structures built of bamboos and woods and covered with 'Nipa Palm Leaves.' These widespread destructions of homes are now seen among the locals as yet another attempt of cleansing their (i.e. Rohingya) population through systematic displacement of the people.
The order to demolish homes had only been imposed on the Rohingya community, not on the Rakhine Buddhist community.
[Edited by M.S. Anwar]
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MHADA Admit Card 2017 2018 Download Maharashtra Junior Clerk Exam Hall Ticket 2017 mhada.maharashtra.gov.in
MHADA Admit Card 2017 2018: Maharashtra Housing and Areas Development Authority will going to conduct the MHADA Jr Clerk exam 2017 which will be held at the various examination center in Maharashtra state. Applicants who had applied the MHADA recruitment 2017 before the closing date of the application submission. Now they can download the MHADA Junior Clerk admit card 2017 through the official site. Our Indrajobinfo.co.in website has issued the direct link for the MHADA Jr Clerk hall ticket 2017. So the aspirants can get their MHADA Junior Clerk exam admit card 2017 by clicking given link.
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The candidates who are seeking the latest govt jobs in Maharashtra had applied the MHADA Jr Clerk recruitment can download the MHADA Junior Clerk exam hall ticket 2017. Many of the candidates are eagerly waiting for download the MHADA admit card 2017. You can get their MHADA Jr Clerk call letter by using given link provided below.
Facebook Like Page: Latest Jobs Updates
MHADA Exam Admit Card 2017:
Eligible candidates can download the MHADA Junior Clerk exam admit card 2017 from the official site mhada.maharashtra.gov.in. Once you download the MHADA call letter 2017 and then you will check the following details like name, date of exam, exam venue and other information. Suppose you found any misspells on the MHADA Maharashtra admit card 2017. So you will contact the issuing authority of the recruitment. They will resolve your problem.
MHADA Jr Clerk Hall Ticket 2017:
Now days candidates are searching the MHADA admit card 2017 on official site. If the official site has publish the MHADA Junior Clerk call letter 2017. We will update the direct link for the MHADA Jr Clerk exam hall ticket 2017 in Indrajobinfo. There you can also check the latest jobs in India 2017. We provide the guidelines to the candidates how to download the MHADA hall ticket 2017 from the official portal.
- First of all click the given link.
- You will redirect to the official page.
- Click on the MHADA Junior Clerk hall ticket 2017.
- Put your registration number and password.
- Call letter will be shown on your screen.
- Take the print out in A4 size for exam purpose.
Important Dates:
Date of MHADA Jr Clerk Exam 2017:
Download Link: MHADA Junior Clerk Exam Admit Card 2017.


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Monday September 28, 2015 CrossFit Stapleton- Denver, CO
New class announcement!
We are VERY excited to announce a new class to the CFS Schedule…
This is a barbell club that will meet twice a week to lift some heavy weights and learn new accessory movements pertaining to lifting and gymnastic, lead by Emilio!
We will meet every Tuesday and Thursday at 6:30pm! This class will be 60-90 minutes… We will be following Hot Dogs & Cupcakes Strength Programming.
Unlimited members get access to this program for no extra charge, 3x/week members can add this bonus class on for an addition $20/mo charge.
We will also be inviting non-CFS members to join this class for a $75/mo charge.
Please email email@example.com with questions.
This class begins Tuesday October 6th!!!
A. Every 4 minutes, for 20 minutes (5 sets) of:
Clean x 18.104.22.168 (rest 10 seconds between singles)
Rest 30 seconds
Strict Handstand Push-Ups x max reps
Note weight used and reps achieved.
B. Three rounds for time of:
15 Deadlifts (155/105 lbs)
12 Hang Power Cleans (155/105 lbs)
9 Front Squats (155/105 lbs)
6 Jerks (155/105 lbs)
A. Every minute, on the minute, for 20 minutes (4 sets of each):
Minute 1 – Deadlift x 6-8 reps @ 30X1
Minute 2 – L-Seated DB Press x 6-8 reps @ 20X1
Minute 3 – Russian Step-Ups x 10 reps on Left Leg
Minute 4 – Russian Step-Ups x 10 reps on Right Leg
Minute 5 – Supine Leg Lowering x 6-8 reps @ 5010
B. Three rounds for time of:
Row 300 Meters
20 Kettlebell Swings
10 Goblet Squats
Use the same kettlebell for both movements.
Interval WOD- 4 Sets:
Row 500
40 Air Squats
30 sit ups
20 push ups
10 pull ups
rest = work


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HAHA! Just kidding. Wyatt is my youngest nephew and he just turned two years old. He's not a monster at all - he's actually a total sweetheart. But then again, so are these three characters:
I know I should have put ribbon into the little slots on the happy birthday tag, but none of the ribbons I had looked right. So I just stapled that sucker right on there. Wyatt does not have discriminating taste, luckily for me! (Just another reason why I love him so very much.)
Tune in tomorrow for another "bird-day" card. I hope that doesn't ruin the surprise...
Have a great evening!
Love Shelley


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Production of the Mustang started in Dearborn, Michigan on March 9, 1964 and the car was introduced to the public on April 17, 1964 at the New York World's Fair. It is Ford's third earliest nameplate presently in production. Initial sales projections projected less than 100,000 devices for the very first year. This mark was surpassed in three months from rollout. Another 318,000 would be offered during the model year (a record), and in its very first eighteen months, more than one million Mustangs were developed.
Buy Classic Ford Mustangs
All these were VIN-identified as 1965 designs, but a number of changes were made at the traditional opening of the brand-new model year (start August 1964), including the addition of back-up lights on some models, the introduction of alternators to replace generators, and an upgrade of the V8 engine from 260 cu in to 289 cu in. When it comes to at least some six-cylinder Mustangs fitted with the 101 hp 170 cu in Falcon engine, the rush into production consisted of some uncommon peculiarities, such as a horn ring bearing the 'Ford Falcon' logo design beneath a trim ring emblazoned with 'Ford Mustang.' These attributes made enough distinction to necessitate classification of the 121,538 earlier ones as "1964 1/2" model-year Mustangs, a difference that has actually sustained with purists. Mustangs For Sale in Glenwood GA are easy to find now with online shopping.
The 1973 OPEC oil embargo brought with it more strict pollution laws and the desire for fuel-efficiency. This was likewise the very first time in vehicle history that the Huge 3 were having to compete with imports. The brand-new Mustang II was smaller sized than the first variation but, due to the additional devices needed to satisfy new US emission and safety policies, was heavier than the very first design. Efficiency was reduced and in the first year just 385,993 cars were sold.
2nd generation Mustangs for sale in Glenwood GA were available between 1974 and 1978 and offered in a coupe and hatchback version. 1979 the Mustang was revamped a third time. This time it was based on the larger Fox platform and the interior was really big enough for four individuals. The trunk was larger and so was the engine bay. The body designs readily available consisted of a coupe, hatchback and in 1983 a convertible. In 1979 the Third generation Mustang functioned as an Indianapolis 500 speed car and Ford developed 10,478 celebratory replicas. The 3rd generation Mustangs were produced from 1979 to 1982.


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PROVO It was thumbs down for a 650-lot development west of Utah Lake recently, but county commissioners could reverse their Planning Commission when they meet Tuesday to consider the project.
Developer Cole Cannon's West Lake Estates, a temporary name, received a mostly favorable response from the commissioners when he appealed his rejection last Tuesday to rezone 180 acres from agricultural uses to high-density residential. The site is close to the south end of Utah Lake near West Mountain.
Rezoning would precede a 12-year plan to build a new 400-acre community that would have its own recyclable gray water system. A million-gallon water tank buried in the mountain above the community would provide culinary water.
However, the county won't provide any services, except police. A contract would have to be worked out for fire protection, Commissioner Steve White said. The closest town is Payson.
Fire hydrants would be another requirement.
A homeowners association would take care of other services or the project wouldn't get approved, White said.
Homes built in the community could have personal windmills or solar panels to provide about half the power needed, or homeowners could purchase green credits for power that would come from hydroelectric or other wind power sources, Cannon said.
"I want (this) to be the first (self-) sustainable community in Utah County," he said.
The development would have home density much greater than the county norm of one home for every five acres. Cannon's average lot size is .68 of an acre. The smallest would measure 7,000 square feet and the largest estate lots would top one acre, Cannon said.
Once the community's population reached 100 it would have to incorporate.
Issues to work through include water. The land has one well now, but more are in the approval stages, he said.
The county wants to preserve agriculture, but the site for Cannon's project isn't favorable to farming or grazing. It could negatively affect other farms in the area, officials said.
Once the county rezones the property it has no ordinance to enforce the development plan, White said. However, Cannon said, the zoning requirements are stringent.
E-mail: [email protected]


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In 1976, having completed two praiseworthy projects for Spanish filmmaker Carlos Saura, Geraldine Chaplin journeyed to Spain to appear in Enrique Braso's In Memoriam. Her contribution to the film is secondary to that of Jose Luis Gomez, who play Julio...more
All In Memoriam cast + crew photos
Movies.com, the ultimate source for everything movies, is your destination for new movie trailers, reviews, photos, times, tickets + more! Stay in the know with the latest movie news and cast interviews at Movies.com.
Copyright © 2019 FANDANGO MOVIES.


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JA of Central Virginia Executive Committee Members
Sarah C. Martin — Chair
PwC, LLP
Frasier W. Brickhouse, II — Vice Chair
Tredegar Corporation
Andrew Carington — Secretary
Co-Founder at CelsiusGC, PLC
Benny Bowman — Chair, Board Governance
Worth Higgins & Associates
Helen Dow — Chair, Finance & Audit
Goodwill of Central and Coastal Virginia
Duane Williams — Chair, Fundraising
UPS Freight
Amy Miller — Co-Chair, Mission Advancement
Vaco Richmond
Angela Roisten — Co-Chair, Mission Advancement
Virginia Credit Union
Simon Hodges — Past Chair
Dominion Energy
Valerie Heinz — At Large
CarMax, Inc.
Pat Collins — At Large
TowneBank Richmond
JA of Central Virginia Board of Directors
Jillian Avey
Markel Corporation
Larry Brown
Horton International
Katie Burnett
Wells Fargo
I. Lee Chapman, IV
Davenport & Company, LLC
Ron Carey
Tilt Creative+Production
Brian Combs
Chris Crowe
Lumber Liquidators
Terrye Enslow
Luck Companies
Robert P. Fisher
Danielle Fitz-Hugh
Chesterfield Chamber
Kyle Harkrader
Mitchell W. "Mitch" Hassell
Danielle Gilbert
Manifest in You Consulting, LLC
Kristin Henshaw
Lee Hannah
Cigna HealthCare
Matthew Head
Mayer Electric Supply
Canon Hickman
Equity Concepts
Kathy Houghtaling
Federal Reserve Bank of Richmond
Robert A. Huebner
Bluestone Process Dynamics, LLC
Charles Kemp
Williams Mullen
Brian Johnson
Genworth Financial
Stephanie Karfias
SunTrust Mortgage
Brian C. Lange
South State Bank
Toby R. Leslie
Peter John Ramsey, PhD.
Pfizer Consumer Healthcare
Christopher V. Lucy
Benjamin Ross
M&T Bank
Mary G. Morris
Virginia529 College Savings Plan
Walter Preston
State Farm
Ana Reilly
Gumenick Properties
Matthew Schaaf
Gerard Shaia
Costen Floors
John Sliman
Leah Walder
Leah Walder Interiors
Paul Ward
Bank of America
Theresa Wills
Capital One
JA of Central Virginia Board of Directors Emeritus Members
Jonathan B. Macdonald, Esq.
Joseph C. Ramage
JA of Central Virginia Education Advisory Board
Jason Kamras
Richmond Public Schools
Dr. Michael B. Gill
Hanover County Public Schools
Dr. Eric Jones
Powhatan County Public Schools
Dr. Amy E. Cashwell
Henrico County Public Schools
In Transition
Chesterfield County Public Schools
Dr. Dave Myers
New Kent County Public Schools
Dr. Marcus J. Newsome
Petersburg Public Schools
Dr. Jeremy Raley
Goochland County Public Schools
JA Young Professionals Board
Vanessa Hampton — Chair
Tim Hendricks — Secretary
Capital One
Mack Frost — Vice Chair
Federal Highway Administration
Annie Cai Larson — Marketing Chair
Genworth Financial
John Pendleton — Immediate Past Chair
Scott Insurance
April Auger-Perrin
Jamie Billingsly
Dominion Energy
Ryan Angus
UPS Freight
Allen Bowman
Federal Reserve Bank of Richmond
Erin Cheeley
Luck Companies
Leesa Christian
Markel Corporation
Angel Clarke
Bank of America
William Clinton
Bon Secours
Shannon Early
Carter French
Education Advisory Board
Rachel Griggs
Cherry Bekaert
Bonnie Hamilton
The Brink's Company
Dollie Kelly
C&F Bank
Caitlin Kilcoin
Education Advisory Board
Justin Lassiter
Kaleo Pharma
Jennifer Likander
Markel Corporation
Andrew Miller
Workshop Digital
Lauren Minnick
Brittany Poore
Bon Secours Virginia Health System
Sarah Schneider
Sara Stone
Logan Tinder
LaKrisha Watkins
Dixon Hughes Goodman
"Junior Achievement has given me a sense of what adults go through with budget issues."
"Junior Achievement reinforced concepts for me to remember later in life."
"I thought the experience was amazing. The presentation was unlike anything I've seen."
"I liked how the Junior Achievement volunteer explained his job to us."


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Two Police Department Recruiting Videos Couldn't Be More Different
The city budgets are always a topic of discussion around the Tri-State. Police and Fire departments seemingly are always being asked to do more with less, but they are consistently recruiting good candidates to fill positions as they become available through attrition or expanded budgets that allows more to be hired. Police departments generally recruit a couple of times a year and the recruiting process can be tricky, especially if you are using a recruiting video like Decatur, Georgia and Newport Beach, California, where I was born and raised.
The below videos are polar opposites of each other with Decatur showing a quiet laid back approach and using an empathetic attitude in their video showing an officer riding a bike and the benefits of living in Decatur.
Newport Beach takes an approach that is the exact opposite, taking a "Do you have what it takes?' approach in it's video with guns blazing, recruits in the academy and a lot of bad guys being taken down. This is a very big surprise for me.
I was raised in Newport Beach and even grew up in that very police department going from Police Explorer to Cadet in my time there. When I was there, community relations were first and foremost and recruiting involved more the benefits of living and protecting a beautiful, coastal community. The department I see in the below video is not at all what I remember, but each department takes its own approach when recruiting.
I am certainly not saying Newport Beach is wrong in their approach, nor am I saying Decatur is right with theirs. It is just very interesting to see how differently departments recruit and the message they send. See both videos below and judge for yourself.


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Most small nucleolar RNAs (snoRNAs) fall into two families, known as the box C/D and box H/ACA snoRNAs. The various box elements are essential for snoRNA production and for snoRNA‐directed modification of rRNA nucleotides. In the case of the box C/D snoRNAs, boxes C and D and an adjoining stem form a vital structure, known as the box C/D motif. Here, we examined expression of natural and artificial box C/D snoRNAs in yeast and mammalian cells, to assess the role of the box C/D motif in snoRNA localization. The results demonstrate that the motif is necessary and sufficient for nucleolar targeting, both in yeast and mammals. Moreover, in mammalian cells, RNA is targeted to coiled bodies as well. Thus, the box C/D motif is the first intranuclear RNA trafficking signal identified for an RNA family. Remarkably, it also couples snoRNA localization with synthesis and, most likely, function. The distribution of snoRNA precursors in mammalian cells suggests that this coupling is provided by a specific protein(s) which binds the box C/D motif during or rapidly after snoRNA transcription. The conserved nature of the box C/D motif indicates that its role in coupling production and localization of snoRNAs is of ancient evolutionary origin.
Biogenesis of eukaryotic RNAs involves maturation of primary transcripts and targeting of product RNAs to sites where they function. Clearly, these two fundamental processes are coordinated in the cell, since precursor RNAs are usually found at the sites of synthesis, while only mature RNAs accumulate at the sites of function. This situation is believed to result from two consecutive events: (i) initial assembly of a macromolecular complex which is required for RNA maturation; and (ii) targeting of mature RNA to its site of function.
During the maturation of mRNA, precursors are retained near their site of synthesis (Singer and Green, 1997, and references therein). Subsequently, signals provided by the mature RNA direct it to the cytoplasm (Izaurralde and Mattaj, 1995, and references therein). These two basic processes have been shown to be coupled as, in some cases, efficient targeting (e.g. nuclear export) requires prior processing of the RNA (e.g. 3′ end formation) (Eckner et al., 1991; Huang and Carmichael, 1996). This coupling is manifested spatially at the cellular level by localization of the precursor only at the transcription site (Zhang et al., 1994).
Less is known about RNAs which are retained and targeted within the nucleus. The snoRNAs are a good example of RNAs which traffic intranuclearly. SnoRNAs exist in the cell in the form of RNP complexes (snoRNPs), and have been discovered in protozoan, fungal, plant and mammalian cells (reviewed in Gerbi, 1995; Maxwell and Fournier, 1995; Smith and Steitz, 1997; Tollervey and Kiss, 1997). Nearly all have been firmly tied to maturation of rRNA, specifically cleavage of pre‐rRNA and synthesis of modified nucleotides (see also Maden, 1996; Peculis and Mount, 1996; Bachellerie and Cavaille, 1997). Based on conserved structural features, snoRNAs are now classified into three subsets (Balakin et al., 1996; Ganot et al., 1997; reviewed in Smith and Steitz, 1997; Tollervey and Kiss, 1997). Two of the subsets define large families known as the box C/D and box H/ACA families. These families account for all but one snoRNA; the exception is the snoRNA known as MRP (Maxwell and Fournier, 1995; Tollervey and Kiss, 1997)
Members of the box C/D snoRNA family, which are the subject of the present report, possess characteristic sequence elements known as box C (UGAUGA) and box D (GUCUGA). Most box C/D snoRNAs contain long (>12 nucleotides) sequences complementary to rRNA, which, in conjunction with adjoining box D or D′ elements, target rRNA nucleotides to be ribose methylated (reviewed in Bachellerie and Cavaille, 1997; Smith and Steitz, 1997; Tollervey and Kiss, 1997; see also Cavaille et al., 1996; Kiss‐Laszlo et al., 1996; Nicoloso et al., 1996; Tycowski et al., 1996). A few box C/D snoRNAs are required for cleavage of pre‐rRNA, and this essential function does not appear to be related to methylation.
The coding sequences for the box C/D snoRNAs can be found in pre‐mRNA introns or in mono‐ or polycistronic snoRNA genes. Thus, in the early stages, transcription and processing of individual snoRNAs differ according to the nature of the coding unit and, hence, the primary transcript. However, the final maturation steps in each case are believed to be similar, and involve exonucleolytic trimming of unprotected RNA ends (reviewed in Tollervey and Kiss, 1997). When the snoRNA possesses a 5′ cap, this structure protects the 5′ end from degradation. In all cases, the box C and D elements function in both RNA stabilization and maturation. In various organisms, mutations in these elements affect snoRNA accumulation (Baserga et al., 1992; Huang et al., 1992; Peculis and Steitz, 1994; Terns et al., 1995; Caffarelli et al., 1996; Cavaille and Bachellerie, 1996; Watkins et al., 1996; Mereau et al., 1997; Xia et al., 1997; Samarsky and Fournier, 1998). Boxes C and D are usually flanked with inverted repeats, and the base pairing of these segments is also required for RNA accumulation. On this basis, the two boxes and the neighboring helix have been proposed to define a vital structure called the box C/D motif. This motif is believed to be a recognition signal for one or more proteins, which provide metabolic stability of RNA and define the end points in its processing (see also Tyc and Steitz, 1989; Ganot et al., 1997). Evidence that the putative box C/D motif‐binding protein(s) is common and conserved comes from studies showing that: (i) injection of a box C/D snoRNA into Xenopus oocytes inhibits accumulation of other box C/D snoRNAs (Terns et al., 1995; Watkins et al., 1996); and (ii) box C/D snoRNAs from distantly related organisms are stabilized and processed in yeast and animal cells (Li and Fournier, 1992; D.R.Newman, J.C.Liu, M.J.Fournier and E.S.Maxwell, unpublished data).
Another, more mysterious aspect of box C/D snoRNA biogenesis is the process by which these RNAs are localized within the nucleus. The snoRNAs are highly enriched in the nucleolus, as the name implies; however, the mechanism and signals responsible for this precise localization are unknown. This situation reflects our general lack of knowledge about intranuclear RNA trafficking. Indeed, although most eukaryotic RNAs travel through the nucleus, very few examples of intranuclear RNA targeting have yet been analyzed (Jacobson et al., 1995, 1997).
The investigation described here was undertaken to gain new insight into the principles of box C/D snoRNA biogenesis. Specifically, we wished to define snoRNA elements sufficient for nucleolar targeting, and to assess the potential inter‐relationship between the processes of snoRNA localization and production. Experiments were conducted in both yeast and mammalian cells, using a natural yeast snoRNA and a variety of artificial variants.
Our study was initiated by analyzing the biogenesis of the U14 box C/D snoRNA (Figure 1) in the yeast Saccharomyces cerevisiae. U14 is essential for processing of 18S rRNA (Zagorski et al., 1988; Li et al., 1990; Liang and Fournier, 1995), and is involved in methylation of rRNA (Kiss‐Laszlo et al., 1996; Dunbar and Baserga, 1998). Homologs of U14 have been identified in >20 organisms, including fungi, plants and animals (see the electronic database by Zwieb, 1997). The U14 coding sequence is found in various genomic arrangements in different eukaryotes, including mono‐ and polycistronic transcription units and within introns of protein genes (Zagorski et al., 1988; Liu and Maxwell, 1990; Leverette et al., 1992; Leader et al., 1994; Xia et al., 1995; Samarsky et al., 1996; Dichtl et al., 1997; Leader et al., 1997; Petfalski et al., 1998). In S.cerevisiae, U14 is encoded ∼70 bp downstream of another box C/D snoRNA, snR190. Both snoRNAs are co‐transcribed, and the mature molecules are derived by endonucleolytic cleavage of the dimeric precursor, followed by exonucleolytic trimming (Petfalski et al., 1998). The conserved nature of U14 and the fact that it is produced from different types of precursors make this snoRNA particularly attractive for defining the general principles involved in biogenesis of box C/D snoRNAs.
The box C/D motif provides metabolic stability and nucleolar localization of snoRNA in yeast
Previous mutagenesis studies of yeast U14 yielded stable variants for an assortment of deletions and substitutions that collectively spanned nearly all of the region between boxes C and D (Jarmolowski et al., 1990). These results indicate that the internal segment does not contain information required for snoRNA production. However, in no case was the effect of a clean internal deletion or substitution examined. To resolve this issue, we created four gene constructs in which artificial sequences replaced the 104 bp segment that normally lies between the box elements (pRA1–pRA4; Figure 2A). The artificial segments were 47, 54, 120 and 228 bp in length. After expression in yeast, each new gene produced a single stable RNA of the expected size, corresponding to the artificial portion, boxes C and D and a terminal stem (∼69, 76, 142 and 250 nucleotides; Figure 2B, see also Figure 1A). The artificial RNAs accumulated at levels similar to that of natural U14 expressed in the same genetic context (control data are not shown). Double point mutations in either box C or box D, known to abolish yeast U14 production (Huang et al., 1992), blocked accumulation of the artificial RNAs as well (constructs C* and D*; Figure 2). These results demonstrate that the RNA region between boxes C and D does not contain information required for accumulation and processing, and that the box C/D motif is necessary and sufficient for the stability of the artificial box C/D RNAs.
To assess whether the determinants directing snoRNA accumulation are also involved in intracellular targeting, we examined the localization of an artificial box C/D RNA using fluorescent in situ hybridization microscopy (Figure 3). The nucleoli, which in yeast form so‐called 'crescent bodies', were visualized with a probe specific for natural U14. Strikingly, the same pattern was obtained with a probe specific for the artificial RNA, and double hybridization showed that both RNAs co‐localize. The specificity of the signals was verified by hybridization of cells harboring a plasmid that does not encode any snoRNA (data not shown). These results indicate that the box C/D motif is sufficient to localize RNA to the nucleolus in yeast cells.
The box C/D motif directs snoRNA production and nucleolar localization in mammalian cells
We next asked if the cis‐acting determinants shown to be sufficient for box C/D RNA accumulation and nucleolar localization in yeast play similar roles in mammalian cells. To this end, we analyzed expression of yeast U14 and one of the artificial box C/D RNAs in monkey cells. The artificial RNA contained a 120 nucleotide non‐natural sequence between boxes C and D (from pRA3; see Figure 2). The RNAs were expressed in monkey COS‐1 cells, from the human U6 snRNA gene promoter. The U6 promoter utilizes RNA polymerase III but, in contrast to conventional Pol III promoters, is located upstream of the transcribed region, rather than within. Thus, little constraint is placed on the sequence of the transcript. An added advantage of this promoter is that transcripts are not capped, which eliminates potential complications associated with a 5′ cap structure.
The genes to be tested were cloned into the mammalian pU6+1 expression vector (Bertrand et al., 1997) to yield constructs pU6Y (yeast U14) and pU6A (artificial RNA; Figure 4A). Each new gene contained yeast DNA that normally occurs up‐ and downstream of the coding sequence for mature yeast U14, ∼30 bp on the 5′ side and ∼130 bp on the 3′ side. Precursor RNAs derived from these constructs were predicted to be ∼225 (yeast U14) and ∼250 nucleotides (artificial RNA), based on the assumption that a stretch of seven T nucleotides, located 72 bp downstream of the box D, serves as a termination signal for RNA polymerase III. To avoid the possibility of premature transcription termination of the artificial RNA gene, a run of four T residues immediately upstream of box D was modified by deleting two T nucleotides. The new genes were introduced into monkey cells by transient transfection. Stable RNAs accumulated in each case, and the major products were the same size as the corresponding RNAs produced in yeast (∼130 nucleotides for yeast U14 and ∼155 nucleotides for artificial RNA; Figure 4B). Minor bands corresponding in size to the precursor molecules expected were observed after longer exposure of the Northern blot (data not shown). These results show that the box C/D motif is sufficient for accumulation of box C/D RNAs in phylogenetically distant mammalian cells, as well as in yeast.
Intracellular localization of yeast U14 and the artificial box C/D RNAs was then examined in the monkey cells, using fluorescent in situ hybridization analysis. A hybridization probe recognizing monkey U14 snoRNA was used to visualize the nucleolus. Both, yeast U14 and the artificial RNAs localized to the nucleoli (Figure 5A; data are shown for the artificial RNA). Expression of control RNAs lacking boxes C and D, or possessing only box C, yielded patterns of uniform distribution over the entire nucleoplasm (not shown). This last result verified that the nucleolar localization of yeast U14 and the artificial RNAs is specific and depends on the presence of a box C/D motif. Interestingly, a higher resolution analysis revealed that the artificial RNA localizes in a sub‐nucleolar compartment(s), which shows partial, but not complete overlap with natural monkey U14 or the nucleolar protein fibrillarin (Figure 5B; data for fibrillarin are not shown). This pattern suggests that localization of snoRNAs within the nucleolus is influenced by RNA structural elements in addition to the terminal box C/D motif.
The distribution patterns of the experimental RNAs also showed the presence of sharp, dot‐like signals in the nucleoplasm (see Figure 5A). These dots could correspond to RNA transcription sites (see below), but also to coiled bodies, since at least one box C/D snoRNA, U3, has been found in coiled bodies of mammalian and plant cells (see Jimenez‐Garcia et al., 1994; Raska, 1995; Olmedilla et al., 1997). The latter suggestion was examined by double in situ hybridization with probes for the test RNAs and antibodies against coilin, a specific protein component of coiled bodies (Figure 5C; data are shown for the artificial RNA). The results showed that, indeed, yeast U14 and the artificial RNAs accumulate in coiled bodies as well as in the nucleolus.
To determine where transcription of the test snoRNAs occurs in the nucleus, we performed an in situ hybridization analysis of transfected monkey cells with: (i) a probe specific for a non‐transcribed sequence of the plasmid; and (ii) probes used previously which recognize the mature artificial and yeast U14 snoRNA molecules, respectively. The plasmid probe yielded sharp dots in the nucleoplasm in several places. Differences in signal intensity in the various dots are presumed to reflect differences in gene copy number at the respective sites. Although the plasmid could sometimes be found at the edge of the nucleolus, it was always excluded from this region (data for yeast U14 are shown in Figure 6A, upper panel). Double labeling with coilin antibodies did not reveal co‐localization of plasmids with coiled bodies (not shown). Most of the plasmid sites are transcriptionally active, as revealed by double hybridization with the plasmid and mature RNA probes (Figure 6A, upper panel). These results indicate that yeast U14 and the artificial RNAs are initially produced in the nucleoplasm and targeted to the nucleolus post‐transcriptionally.
Taken together, these results demonstrate that, as in yeast, the box C/D motif is sufficient for RNA accumulation and nucleolar localization in mammalian cells. Thus, in mammals, and presumably in yeast, snoRNAs are transcribed in the nucleoplasm and then transported to the nucleolus. Interestingly, in mammalian cells, the box C/D motif also targets RNAs to the coiled bodies.
Processing of experimental box C/D RNAs occurs in the nucleoplasm of mammalian cells
To determine the sites of box C/D snoRNA processing in the mammalian nucleus, we performed a localization analysis of RNA precursors, once again using yeast U14 and the artificial RNA featured earlier. The precursor probe selected hybridizes specifically to a sequence downstream of box D (see Materials and methods). This probe was used in conjunction with the probes used previously for the mature RNAs and the corresponding plasmid DNA. In situ hybridization analyses showed that precursors localized to distinct areas outside the nucleolus (Figure 6A, central panel; results are shown for yeast U14 only, but are identical for the artificial snoRNA). Double in situ hybridizations with the precursor and plasmid probes showed that the precursors localized predominantly to the same regions as the transfected plasmids (Figure 6A, lower panel).
Detection of RNA precursors at the putative transcription sites suggests that processing of immature RNAs takes place in the nucleoplasm, immediately after transcription. It is possible, though less likely, that maturation occurs in the nucleolus after targeting. In this case, processing would have to occur so rapidly that precursors cannot be detected by our assay. An attempt to resolve these two alternatives was made by analyzing the distribution patterns of non‐processed and processed RNAs in monkey cells transfected with replicative plasmids. These vectors occur at a higher copy number and were expected to yield higher levels of precursors. We reasoned that this strategy could allow unprocessed RNAs to be detected at other nuclear locations.
Overproduction of RNAs was achieved when the box C/D RNA genes were expressed from the replicative plasmids pU6Yrep and pU6Arep (Figure 6B; results are shown for yeast U14, but are identical for the artificial RNA). In situ hybridization demonstrated that despite a much higher expression level, the precursor was still detected only at the transcription sites. Interestingly, in cells expressing the highest levels of experimental RNAs, signals were distributed over the entire nucleoplasm, in addition to the nucleolus. This last result most likely reflects saturation of box C/D RNA uptake and/or retention sites in the nucleolus, leading to nucleoplasmic accumulation of RNAs. This view is supported by our observation that natural monkey U14 snoRNA also accumulates in the nucleoplasm of cells overproducing the experimental box C/D RNAs (data not shown). Parallel hybridizations with precursor‐ and plasmid‐specific probes showed that in overexpressing cells, the precursor RNAs localize at the transcription sites, which form a characteristic speckled pattern, and that the remaining population of RNAs in the nucleoplasm represents processed RNAs (Figure 6B).
Taken together, these results argue that in mammalian cells, precursors to the box C/D snoRNAs are produced in the nucleoplasm, processed at or near the sites of transcription and only then are localized to the nucleolus.
The expression and localization results provide new insights into the biogenesis of the box C/D snoRNAs in eukaryotic cells. In particular, the simple cis‐acting box C/D motif was shown to direct intranuclear trafficking of box C/D snoRNAs, from the nucleoplasmic sites of synthesis to the nucleolus. The new results also argue strongly that production and intracellular targeting of the box C/D snoRNAs are tightly coupled.
Principles of intranuclear localization of box C/D snoRNAs
The localization results obtained with the artificial box C/D snoRNAs showed that, both in yeast and mammalian cells, nucleolar targeting is mediated by the the box C/D motif. Since the box C/D motif defines one of the two major snoRNA families, we predict that it is used as a nucleolar localization signal for all box C/D snoRNAs, i.e. for scores of RNA species in various eukaryotes. In this regard, it is similar to the m7G cap and the poly(A) tail or the Sm‐binding site, which define whole classes of RNAs and also function as determinants of RNA localization (see Hamm et al., 1990; Marshallsay and Luhrmann, 1994; Huang and Carmichael, 1996; Lewis and Izaurralde, 1997). It is tempting to speculate that the unknown localization signals in other RNA families might also be shared by all family members. If true, this common property could greatly facilitate identification of such signals in the future. Thus, it is reasonable to expect that the conserved box elements which define the H/ACA snoRNA family also function in RNA targeting, as well as in RNA production.
In mammalian cells, the artificial RNAs were also detected in the coiled bodies. This last finding is consistent with earlier observations of U3 box C/D snoRNAs in the coiled bodies of animal (human HeLa) and plant (olive) cells (Jimenez‐Garcia et al., 1994; Raska, 1995; Olmedilla et al., 1997), and reinforces striking similarities between the coiled bodies and the nucleolus. Indeed, in addition to snoRNAs, both compartments contain common proteins, including fibrillarin, Nopp140, Nap57 and Sp6 (reviewed in Brasch and Ochs, 1992). In light of the recent discovery that a box C/D snoRNA guides methylation of a splicing snRNA (K.T.Tycowski, Z.‐H.You, P.Graham and J.A. Steitz, personal communication), and the fact that coiled bodies also contain splicing snRNAs (see Brasch and Ochs, 1992; Carmo‐Fonseca et al., 1992; Matera and Ward, 1993; Gall et al., 1995), our results raise the fascinating possibility that methylation of splicing snRNAs occurs in coiled bodies.
Detection of the box C/D RNA precursors at the transcription sites, but never in the nucleolus, suggests that processing occurs at or near the sites of transcription, and the RNAs are targeted to the nucleolus as mature‐length molecules. When expressed at high levels, processed RNAs accumulated throughout the entire nucleus, indicating saturation of nucleolar uptake/retention sites. Importantly, in these cells, the precursor RNAs were still confined to the transcription sites, suggesting that precursor processing was not a limiting factor, and that processing occurs in the nucleoplasm during, or soon after transcription.
A general model of box C/D snoRNA biogenesis
Both RNA maturation and nucleolar targeting appear to be mediated by the same RNA structure, the box C/D motif. Because it seems certain that this motif is a protein recognition signal, and because of its small size, it seems likely that: (i) these functions are mediated by the same protein or a small set of core proteins and (ii) both motif‐dependent processes require formation of the same initial complex.
Accordingly, the major principles of box C/D snoRNA synthesis can be summarized in a simple model (Figure 7). Regardless of the nature of the primary transcript and its initial processing events, it will fold in such a way that boxes C and D are brought together to form the box C/D motif. The motif is then rapidly recognized and bound by a nucleoplasmic protein which alone, or with other proteins, protects the snoRNA from degradation by exonucleases that specify the ends of the mature molecule. This initial RNP complex alone, or in conjunction with additional nucleoplasmic factors, is then delivered to the nucleolus. Translocation could occur by active transport or by simple diffusion and subsequent nucleolar binding through common protein components.
Maturation of box C/D snoRNAs can also include: (i) modification of internal nucleotides, about which little is known (see Reddy et al., 1979; Wise et al., 1983; Reddy and Busch, 1988); and (ii) formation of a 5′ trimethylguanosine (TMG) cap. TMG cap formation is believed to result from hypermethylation of the m7G cap normally found in RNA polymerase II transcripts. Interestingly, hypermethylation of the Xenopus U3 and U8 caps occurs in the nucleoplasm, and has been shown to depend on box D and the adjoining terminal stem (Terns et al., 1995). This last finding raises the possibility that hypermethylation also depends on the box C/D motif, i.e. that TMG synthesis is activated shortly after transcription by the same motif‐specific protein(s).
It seems certain that direct binding of a motif‐specific protein(s) and conversion of naked RNA to a core snoRNP complex is a pivotal step in both production and localization of the box C/D snoRNAs. This arrangement provides a reliable and efficient means of integrating these two major aspects of snoRNA biogenesis, since common factors are required for both processes and render them inseparable. Such coupling might be common for other classes of eukaryotic RNA as well. For example, both processing and nuclear export of mRNAs are believed to be affected by some hnRNP proteins and components of the nuclear cap‐binding complex (reviewed in Weighardt et al., 1996; Lewis and Izaurralde, 1997).
In addition to integrating the various aspects of snoRNA synthesis (maturation, stability and localization), there is the intriguing possibility that the box C/D motif also links snoRNA synthesis with function. In particular, this possibility applies to the snoRNAs which guide methylation of rRNA. The guide function is mediated by a long sequence complementary to the rRNA segment to be modified, and site selection depends on a box D or D‐like element (CUGA, box D′) located immediately downstream of the guide sequence. Methylation occurs in the complementary rRNA sequence, precisely five nucleotides from box D/D′. Some guide sequences occur near the 3′ end, immediately upstream of the canonical box D, whereas others occur in the interior of the RNA adjoined to box D′. Some snoRNAs contain guide sequences in both arrangements. Interestingly, we and others noted that snoRNAs with internal guide sequences and box D′ also contain an additional box C‐like sequence (UGAU) at a modest distance downstream of box D′, suggesting that these RNAs have a second box C/D motif, in addition to the canonical one (Kiss‐Laszlo et al., 1998; D.A.Samarsky, unpublished). Results from recent mutational studies have demonstrated that the novel box C‐like element (called box C′) is essential for the methylation reaction, but not for snoRNA accumulation (Kiss‐Laszlo et al., 1998). This finding argues that one or more proteins involved in the methylation function also bind directly or indirectly to the simple box C/D motif, and that snoRNA production and function may be connected at this level. It will be interesting to learn if the motif‐binding protein(s) includes a ribose methylase enzyme.
The strict conservation of the box C/D motif among different snoRNAs in evolutionarily divergent eukaryotes indicates that the principles of box C/D snoRNA biogenesis, in particular synthesis and intranuclear targeting, are universal to all RNAs in this family and, hence, are as ancient as the eukaryotic world itself.
Materials and methods
Yeast strains and mammalian cell lines
All yeast experiments were carried out with the S.cerevisiae haploid strain YSD92 (ura3‐167). This strain was prepared by sporulating strain YS152 (a/α his3Δ/HIS3 his4‐280/HIS4 trp1/TRP1 ura3‐167/URA3 GAL1::U14:HIS3/U14) described previously as a diploid obtained after mating of haploid strains YS133 and E280 (Jarmolowski et al., 1990). Yeast transformants were grown on YNBD (0.67% yeast nitrogen base, 2% glucose) medium at 30°C.
COS‐1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. Transfections were performed by a DNA–calcium phosphate co‐precipitation method (Sambrook et al., 1989), using 10 μg of DNA per 3 cm dish. Cells were fixed 36 h after addition of the precipitate.
Plasmid construction
The artificial snoRNA sequences were derived from Escherichia coli vector pUC18 and newly created plasmids pBl‐B, pBl‐KS and pBl‐C. The latter constructs were prepared from the pBluescript IISK(−) E.coli vector by deleting a BssHII restriction fragment (pBl‐B), deleting a KpnI–SacI fragment (pBl‐KS) or removing a ClaI restriction site (pBl‐C).
Yeast plasmids pRA1, pRA2, pRA3 and pRA4 were prepared from a previously created plasmid pCer (Samarsky et al., 1996). This parental plasmid contains a 1.3 kb ClaI S.cerevisiae genomic DNA fragment with genes for snR190 and U14 snoRNAs, cloned into a ClaI site of yeast vector pRS316. The pRA1–pRA4 derivatives were made by substituting the DNA between U14 boxes C and D with DNA fragments located between and encompassing the universal primer regions of plasmids pUC18, pBl(‐B), pBl(‐KS) and pBl(‐C), respectively. The DNA replacement fragments were prepared by PCR amplification with oligonucleotide primers SD15 (ATTTATGATCACGGTGATGAGGAAACAGCTATGACCAT) and SD18 (CTACAGTATACGATCACTCAGACGTAAAACGACGGCCAGTGA). Oligonucleotides SD49 (TATATATAATTTATGATCACGGTCTTGAGG) and SD50 (AAGATACTACAGTATACGATCACAGAGACG) were used for making pRA1–pRA4 derivatives containing mutant box C (box C*) or box D (box D*) elements.
Plasmid pU6Y and pU6A were obtained by cloning into the pU6+1 vector (Bertrand et al., 1997) the PCR products obtained from plasmids pCer and pRA3 with the oligonucleotides SD24 and SD81 (GTGGGTAATTTGAGTTAACAGATAATATAT). Plasmids pU6Yrep and pU6Arep were obtained by cloning the SV40 origin of replication into the pU6Y and pU6A plasmids. Detailed restriction maps are available upon request.
RNA preparation and Northern blot analysis
Total RNA from yeast cells was isolated using an acidic phenol/glass bead procedure (Kohrer and Domdey, 1991). RNA from mammalian cells was prepared using the guanidine‐HCl method (Sambrook et al., 1989). Northern analysis was performed as described previously (Balakin et al., 1993; Samarsky et al., 1995). Oligonucleotide probes used for hybridization were C106 (CGATGGGTTCGTAAGCGTACTCCTACCGTGG), specific for yeast U14 snoRNA, and SD18 (see above), specific for all artificial RNAs, except RNA expressed in mammalian cells. Oligonucleotide SD121 (GCGTAATACGACTCACTATAGGGCGAATTGGCC) was used to probe artificial RNA expressed in mammalian cells.
In situ hybridization
Mammalian cells were fixed for 10 min at room temperature in 4% formaldehyde, 10% acetic acid, phosphate‐buffered saline (PBS; 100 mM Na2HPO4, 20 mM KH2PO4, 137 mM NaCl, 27 mM KCl, pH 7.4). After two washes in PBS, cells were permeabilized by treatment with 70% ethanol for at least overnight. After rehydration in 2× SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0), 50% formamide, cells were hybridized overnight at 37°C in 40 μl of a mixture containing 10% dextran sulfate, 2 mM vanadyl‐ribonucleoside complex, 0.02% RNase‐free bovine seum albumin (BSA), 40 μg of E.coli tRNA, 2× SSC, 50% formamide, 30 ng of probe. Cells were then washed twice for 30 min at the appropriate stringency (2× SSC, 50% formamide, 37°C for oligonucleotide probes; 0.1× SSC, 50% formamide, 50°C for RNA probes). When required, slides were then processed for immunofluorescence. Digoxigenin‐labeled probes were detected with sheep anti‐digoxigenin antibodies (1/200, Boehringer Mannheim), and then with donkey anti‐sheep antibodies conjugated to fluorescein (1/150, Sigma). Slides were incubated for 1 h at 37°C in 2× SSC, 8% formamide, 2 mM vanadyl‐ribonucleoside complex, 0.2% RNase‐free BSA, and washed twice for 15 min in 2× SSC, 8% formamide at room temperature.
Yeast cells were prepared for in situ hybridization as previously described (Long et al., 1995), except that cells were fixed for 10 min at room temperature by addition of 10 ml of 2% formaldehyde, 5% acetic acid, to 45 ml of culture medium. Hybridization was performed as for mammalian cells.
Slides were mounted in 90% glycerol, PBS, 1 mg/ml p‐phenylendiamine, 0.1 μg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI).
Image acquisition and processing
Images were captured using a CellScan system (Scanalytics, Fairfax, VA), with a CH250 CCD camera (Photometrics, Tucson, AZ), mounted on a Provis AX70 epifluorescence microscope (Olympus, Melville, NY). Except for Figure 6B, where only a single plane was recorded, red‐ and green‐filtered images were captured every 200 nm on the z‐axis, for a total of 20–25 images, and were then deconvolved using EPR (exhaustive photon reassignment) software (Scanalytics). A single median plane was recorded for blue‐filtered images. Restored three‐dimensional images were observed to select one or a few consecutive planes which were projected onto the z‐axis. The resulting two‐dimensional images were then scaled so that the signal occupies the full dynamic range of light intensities, pseudocolored and superimposed.
Preparation of probes for in situ hybridization
The probes used to detect natural monkey snoRNAs were amino‐modified DNA oligonucleotides of the following sequences: T*ATCCAAGGAAGGT*AGTTGCCAACAT*AAGACTTTCTGGT*GGAAAC TACGAAT*T for U14 and T*CTTCCTCGTGGTTTT*CGGTGCTCTACACGT*T for U3 snoRNAs (the amino‐modified T nucleotides are marked with asterisks). These oligonucleotides were synthesized and labeled with Cy3 (U14) or Cy2 (U3; Amersham), as previously described (Kislauskis et al., 1993).
All other probes were RNAs synthesized in vitro with T7 or T3 RNA polymerase. The DNA templates were: PCR products encompassing the RNA coding sequence for the artificial box C/D RNA and yeast U14 (yielding probes of 130 and 120 nt, respectively); a plasmid containing the yeast U14 transcribed sequences 3′ to box D for the precursor probe (linearized to yield an ∼130 nucleotide transcript); and pBluescript SK(+) deleted of its polylinker for the plasmid‐specific probe (linearized with PvuII to yield an ∼230 nucleotide probe).
Except for the precursor probe, the RNA probes were labeled with a novel procedure. This protocol was developed to allow for high incorporation of the fluorescent label (typically 5–10%, which is 5–10 times higher than can be obtained by labeling during transcription), and for quantitative studies, since it renders the use of antibodies for signal amplification unnecessary. It was developed as a versatile, low‐cost alternative to amino‐modified synthetic oligonucleotides (Kislauskis et al., 1993), and was derived from an existing protocol used to label RNA for microinjection (Wang et al., 1991). Details of the procedure are available on the web site http://singerlab.aecom.yu.e… Briefly, RNAs were synthesized using amino‐allyl UTP instead of UTP during transcription. Transcription reactions were phenol extracted, RNA was precipitated, resuspended in 1× SSC, and unincorporated nucleotides were removed by gel filtration (1× SSC‐buffered P30 micro‐spin column, Bio‐Rad). RNA was again ethanol precipitated and resuspended in water. Labeling was initiated by mixing 4–50 μg of RNA (depending on the final specific activity desired), resuspended in 70 μl of 0.1 M NaHCO3 buffer, pH 8.8, with one vial of Cy3, or 1 mg of Oregon green 488 (Molecular Probes) resuspended in 30 μl of dimethylsulfoxide (DMSO). Labeling was performed for 48 h in the dark, at room temperature, with occasional vortexing. Unreacted dye was removed by two rounds of ethanol precipitation and washing. Specific activity of the probes was calculated by absorption spectroscopy.
The probes for the artificial RNA and yeast U14 were labeled with a specific activity 20 times lower than for the plasmid probe. This ensured that these probes detected RNA preferentially and not the plasmid. Indeed, a labeled plasmid probe of similar specific activity gave signals much fainter than the ones detected with the artificial and yeast U14 probes (data not shown). The probe for the precursor was labeled with digoxigenin‐UTP during transcription. This probe detected predominantly RNA, not the plasmid, since an antisense precursor probe failed to give signals (data not shown).
We thank Thomas Meier for the gift of coilin antibodies and many helpful discussions, Pascal Chartrand and Roy Long for help in conducting in situ hybridization in yeast, and Elizabeth Furter‐Graves for editorial help. Research by D.A.S. and M.J.F. was supported by NIH grant GM 19351 and NSF grant MCB‐9419007, and that by E.B. and R.H.S. by NIH grants GM54887 and GM 57071.
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