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A River Runs Through…the Home
The latest high-end water features go well beyond yesterday's backyard pool; today's homeowners are building lazy rivers, reef aquariums, and water slides.
When Los Angeles developer Frank Damavandi set out to build his modern Zen home in Santa Monica, Calif., he wanted water close by, so he built one block from the beach. But he soon realized that the Pacific Ocean wasn't enough.
To satisfy his yen, he spent $1.5 million to bring water onto his half-acre property. He built a 7-foot-high waterfall along 70 feet of the property line to separate it from the road. Then, he added four koi ponds in the garden, connecting them with a smaller waterfall and a creek. Finally, he installed a saltwater infinity pool and Jacuzzi right up against his house, so the kitchen's glass floor extended over the pool, creating a swim-up bar.
"I've lived at the beach most of my life, and I find water very meditative and soothing," says Mr. Damavandi. "I wanted to live in a house that really revolved around water and its calming effects."
Water features, once a second thought when it came to residential design, are now moving to the forefront of home construction. And the latest high-end water features go well beyond yesterday's backyard pool; today's homeowners are building lazy rivers, reef aquariums, man-made mountains with water slides and massive moats. Still, builders say some homeowners aren't prepared for the sticker shock when installing these features or the overhead of maintaining them.
Costs for water features can run high; designers and architects who specialize in building them say the features can rival the price of a house, topping $1 million in some cases. Los Angeles architect David Montalba says that he had a client who planned to install $800,000 worth of water features in a home, only to change his mind at the last minute because of the cost.
Maintaining such features can be a pricey proposition, too: Mr. Damavandi says that he pays more than $200,000 in yearly upkeep, and that his monthly water and power bill runs about $7,000—so high that the Los Angeles Department of Water and Power sent a supervisor to his house soon after he completed building to make sure nothing was amiss. The intense upkeep the property requires is, in part, why he recently decided to sell the house, listing it for $18.75 million.
"Good architecture has always used water," says Charles Sieger, a Miami-based architect who specializes in luxury high-rises. "A wider audience is finally waking up to that." Mr. Sieger's own house in Miami, surrounded by an 18 million-gallon moat, is accessible via a timber-surfaced bridge.
Mr. Montalba says about half of his clients request water features today, a stark rise from the handful of people who were interested 10 years ago. "Back then, it was just a couple of our most adventurous clients who wanted water in their homes," he says. "Today, everyone seems to want a reflecting pool or a fountain or both."
Water features have a practical purpose as well: They mask unpleasant sounds like traffic or noisy neighbors. Sam Johnson, a landscape contractor in Atlanta, says that, because of the additional uses, he's now building about four times as many residential water features as he did five years ago. "Not everyone can live on a beautiful, placid lake, but we can bring the placid lake to them," Mr. Johnson says.
That's the approach that Mr. Sieger, the Miami architect, took. Studying French châteaus in college, Mr. Sieger had always dreamed of living in one. So he spent five years building a 20,000-square-foot castlelike home surrounded by a 12-foot-deep, 5-acre moat dug out of an abandoned tomato field. Steppingstones carved from the natural stone base of the lake lead to two concrete gazebos, which flank the seven-bedroom, seven-bathroom house.
Mr. Sieger stocked the moat with koi, geese and swans; ducks, egrets and turtles have migrated to the site from the nearby Everglades. His children and grandchildren now canoe throughout it. The property also has a series of waterfalls, fountains, canals, a lagoon, a koi pond and a swimming pool. There are four freshwater wells on site, including two for domestic purposes, so Mr. Sieger has no water bill to speak of. "That's the beauty of Florida," he says. "You can buy property on the water, or you can just dig and make the water appear instead."
Eugene Lochman, who runs a pool business in the Dallas area, says that he has seen spending on pools and water elements triple since 2008. "Five years ago, it was unheard of for people to drop $120,000 on a nice pool and water feature for their house. But now, it's common," he says. "Rather than go on an elaborate trip, people are investing in their backyards."
Mr. Lochman is one of those people, too. A few years ago, he and his wife, Angie, spent about a quarter of a million dollars—more than a third the cost of their home—building a 287-foot lazy river. The meandering current lets people float around their Prosper, Texas, property or, if they want, they can paddle from the river into a large swimming pool. There's also a man-made cave and multiple waterfalls. The whole water feature requires about 45,000 gallons of water to fill and two 10-horsepower motors to create the current. He can operate the entire apparatus with a remote control.
In Draper, Utah, Ralph and Amanda Dlugas live in a home that was built over a century-old waterfall. A 7-foot-wide ravine bifurcates the 8,000-square-foot house, which is connected with a causeway. There's a man-made mountain in the backyard, complete with two water slides and 11 cascading waterfalls, which spill into a large 75,000-gallon pool and a smaller kids' pool. Eleven different pumps recirculate water through the mountain, which includes a grotto area with a television, couch and bar.
Cleaning such an intricate water feature can be tricky, however. "Frankly, maintenance is a nightmare," says Ms. Dlugas. "We have to get a scuba diver to clean the pool every week," she adds, noting that a green sludge forms on the rock when it's not regularly cleaned. The Dlugases pay about $5,000 a year in cleaning costs, and another $30,000 resealing the mountain last year to prevent cracks from ice and snow.
Aquariums are another water feature that developers say has become popular with homeowners because they can be an inexpensive, low-maintenance way to decorate a home—or give it a unique twist.
Several years ago, Bill Wann, an entrepreneur who lives on a 7½-acre property in Bristol, Wis., built what he calls the world's largest privately owned reef aquarium: a 24-foot-long, 20,000-gallon tank he personally constructed from concrete with acrylic windows. The tank has eels, catfish, stingrays and coral in addition to saltwater fish, such as angelfish and tangs. "I really built and designed the house around the fish tank, which came first," he says. "The whole idea of the house is to sit in the living room and watch the fish, but it's more like they watch us. If you move, they swim through the tank to follow you. They also watch the TV."
Because aquariums give off a tremendous amount of humidity, Mr. Wann sealed the room behind the aquarium with plastic and installed a dedicated ventilation system for the tank. "My last aquarium gave off so much humidity that it ate away the ceiling joists and the roof almost collapsed. I learned my lesson," he says.
The cost and availability of water has increasingly come into play as these features grow more popular. Developers are building in a more eco-conscious manner, constructing homes in areas where well water is available and incorporating water-saving plants into the landscape.
"In many parts of the country, water is absolutely more valuable than gold," says fish habitat and water consultant Shannon Skelton, who runs CFI Global Fisheries Management, a company that helps landowners manage their water assets. In recent years, Mr. Skelton says he has worked with a growing number of clients who have purchased large tracts of land, often thousands acres, that don't come with enough water rights, so they end up having to purchase water elsewhere—at a serious premium.
In some homes, water features can be actually money-saving ventures. Landscaping in dry climates requires a lot of hydration, but a lawn made of water itself demands very little.
That's what Tommy Isola, a 35-year-old commercial real-estate broker in Las Vegas, discovered when he moved into an 8,000-square-foot home with 26,000 gallons of water features, including a large pool, waterfalls and a koi pond.
"My water bill is a fourth of what it would be if I had this area covered with grass," says Mr. Isola. "Water is expensive in Vegas, but to my surprise it's much cheaper to have a backyard of water than a backyard of grass."
To be sure, there can be such a thing as too much water. Frank McKinney, a real-estate developer, says he regrets the number of water features he put into a 15,000-square-foot spec house he built several years ago in Palm Beach County, Fla. He says it took about 15 months to find a buyer for this property, which finally sold for $22.7 million two years ago.
Named Acqua Liana, the 1.6-acre estate sits on a piece of land between the Intracoastal Waterway and the Atlantic Ocean and features a "water floor" and "water walls." A 3-inch-thick piece of bulletproof glass covering a pool functions as the floor of the home's foyer. With the touch of a button, water cascades down the foyer's 24-foot oceanfront windows, creating sheeting water walls.
"If I could do it again, I would tone the water stuff down—some buyers who looked at the house didn't like the feeling that they were walking on water," says Mr. McKinney. "On the other hand, maybe we were just ahead of our time."
Write to Lauren Schuker Blum at email@example.com


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ACHICA > Gifts for Mother's Day > Johnstons Cashmere Lacy Wristwarmer, Light Grey
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Johnstons Cashmere Lacy Wristwarmer, Light Grey
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If you are ordering multiple items, ACHICA aim to send as few deliveries as possible for your convenience – you will only pay one delivery charge.
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Materials: 100% Cashmere
Product Dimensions: 52inches/132cms
Product Code HAD032A3-HA0200
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We hope you will be thrilled with your purchases but in the unlikely event that you want to return something, you can find full return instructions on the back of your delivery note or click here

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The Shwood Francis Acetate Sunglasses feature a rounded silhouette and keyhole bridge for a flattering style on a wide range of face shapes. Combining contemporary style with natural materials, this unisex style is a great choice for those seeking a classic but modern look. The Francis Black & Ebony Sunglasses look best on square, oval, rectangle, diamond, and heart-shaped faces.
Free Shipping on All Domestic Orders
See international and additional shipping information here. Tax is only charged if shipping to California.
If, for any reason, your purchase wasn't exactly perfect, we honor return requests within 30 days from the original purchase date. To be eligible for a return, your item must be unused and in the same condition that you received it in. It must also be in the original packaging. Customer is responsible for return shipping costs. To initiate a return, please fill out the quick form at the bottom of our Returns Page.
Please note that special or custom-made orders are non-refundable, as well as any body care or hygienic products (you wouldn't want to purchase used body wash, right?).
For large adventure and furniture items, such as kayaks or paddle boards, a 20% restocking fee will be incurred as we do team lifts to repackage and put these heavy items back into storage.


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read reviews [3]
Serves 4
Serve this grilled lamb with Mediterranean sides like saffron couscous and grilled zucchini and green and yellow bell peppers. Stuff any leftover lamb in a fresh pita with salad greens, tomatoes, and the chili sauce. The sauce is potent, so a little goes a long way.
In a small bowl, mix 4 tsp. salt with the sugar and spices. Sprinkle all over the lamb, transfer to a large non-reactive dish, and cover; refrigerate for at least 4 hours and up to 2 days.
Heat the oil in a medium (10-inch) sauté pan over medium heat until it's shimmering. Add the onion, jalapeños, and 1 tsp. salt, and cook, stirring occasionally, until the onion softens and becomes translucent, about 8 minutes. Add the garlic, coriander, cumin, and red pepper flakes, and cook, stirring, until the garlic becomes fragrant, about 1 minute. Add the tomato paste, and cook, stirring, until the paste starts to darken, about 1 minute. Add 3/4 cup water and the vinegar; bring to a boil, then simmer until the onion is completely tender, about 10 minutes. Transfer to a food processor and purée. Let cool to room temperature, season to taste with more salt, pepper, and vinegar if needed. Set aside; or transfer to an airtight container and refrigerate for up to three days.
For a gas grill, light the front burner to medium-high and the back burner(s) to medium-low. For a charcoal grill, light a medium-hot fire (400°F) with two-thirds of the coals banked to one side. Clean and oil the grill grates. Grill the lamb over the hotter part of the fire without moving it until it's nicely browned, 6 to 8 minutes. Turn, move to the cooler part of the fire, and cook until an instant-read thermometer inserted into a thicker part of the lamb registers 135°F for medium-rare, 8 to 10 more minutes.
Transfer the lamb to a cutting board, tent with foil, and let rest for 10 minutes. Thinly slice across the grain, transfer to a large platter, and sprinkle with the cilantro. Serve with the sauce on the side.
Photo: Maren Caruso
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text: Is there a way to tell when my subscription is due to expire by reading the mailing label on the front of my T&TH magazine? I renewed some time ago and want to make sure I don't miss any issues. My wife thinks she may have gotten me an extension of my subscription as a gift, but neither of us can find any record of it. Thanks.


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A Touch Of 'Scandal' Is All You Need
Denette Wilford | Posted 01.26.2013 | Canada TV
Read More: Tony Goldwyn, Scandal Review, Shonda Rhimes, Scandal, Kerry Washington, Scandal Tv Show, Canada TV News
There are some TV shows that I can't do anything but watch. While there are some I can have on while I pay my bills/send emails/play Fruit Ninja, there are others that require my undivided attention. You know what I mean. One of those shows is 'Scandal.'


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Tim Hall is in his third season as the Director of Cycling-Head Cycling Coach at Lees-McRae College, and 11th season total coaching collegiate cycling.
Hall comes to the Bobcats from Cumberland University in Lebanon, Tenn., where he served as the head cycling coach for eight seasons. After two seasons at Lees-McRae, he has led the mountain bike team to a third and fifth place finish at nationals, while the cyclocross team has finished in third place twice at nationals. The Bobcats finished the 2013-2014 season ranked fifth overall, and sixth overall in the 2014-2015 season in the Division I Team Omnium Standings.
Hall also has a tremendous background in business, team building, and race promotion. Additionally, Hall has been involved in the community throughout his career, spearheading community service projects such as Ride for Reading and the Wounded Warrior Project. Most recently Hall founded the charity ride Nashville to Natchez for the veterans organization Team Red, White and Blue, which in just two years raised almost $100,000 for American war veterans.
He holds both a Bachelor of Science in Business Administration and a Masters of Business Administration from Cumberland, where he played baseball and earned NAIA All-American honors. He was drafted out of Cumberland in the 31st round of the 1991 MLB draft by the San Diego Padres as a catcher, and played four years of professional baseball for San Diego, Atlanta Braves, and the Corpus Christi Barrcudas.
Coach Hall began bicycling in 1998 and started racing competitively in 2000. He competes as a Category 1 on the road, and Category 2 in cyclocross and mountain. Hall is a native of Nashville, Tenn., and currently resides on Beech Mountain.
What they're saying about Coach Hall
Stuart Lamp, Southeast Regional Coordinator for USA Cycling: "I am ecstatic to see Tim take over as the coach at LMC. Tim will provide excellent leadership, and experience in the sport to take the program to a new level of success."
Dean Peterson, head cycling coach at Marian University: "Tim is one of the pillars and role models of the growing collegiate cycling culture in America. He has worked hard to develop cycling within the comprehensive student-athlete experience through his tremendous work at Cumberland University. I sincerely believe that Tim will bring new strength, stability, and promise to the Lees-McRae cycling team - one of the perennial greats of collegiate cycling."


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by Pamela Anderson
fromFine Cooking
Issue 106
Nothing says summer like a picnic basket full of crunchy, juicy fried chicken. This southern favorite tastes so much better made from scratch than it does coming from a bucket, and it's surprisingly simple to prepare. Once the chicken pieces have been tenderized in a buttermilk marinade, they're tossed with seasoned flour in a paper bag for even coating and extra flavor. Next, in a large, deep, heavy skillet that holds heat well (cast iron, if you have one), the chicken is pan-fried in hot oil—we use a deep fat/candy thermometer to ensure that the temperature is just right for a crunchy, golden crust.
Since the results are guaranteed to be finger-licking good, this may be the only fried chicken recipe you'll ever need.
Brown bag it Shaking the chicken in a paper bag with seasoned flour coats the parts evenly and keeps the mess to a minimum.Fry once This recipe calls for a small chicken so that all of the pieces fit in a large skillet and fry in one batch. It's fine if the pan is crowded.Partially cover Keep the chicken partially covered during the first half of frying; this helps maintain the oil temperature and holds in moisture so the chicken stays juicy and the coating gets crunchy.
Don't toss that frying oil—cool and strain out any crumbs and then refrigerate the oil in a large glass jar for up to six months. You can use it two or three more times.
Tool Kit - Have these kitchen essentials on hand before you start:
• Chef's knife
• Liquid measuring cup
• Measuring spoons
• Dry measuring cups
• Large mixing bowl
• Large doubled brown paper bag
• Large wire cooling rack
• Large rimmed baking sheet
• Heavy-duty 12-inch skillet (preferably cast-iron) with lid (you can use a baking sheet as a lid if necessary)
• Ruler (to measure oil depth)
• Deep fat/candy thermometer
• Tongs
• Instant-read thermometer
Photos: Scott Phillips
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text:
Blade of course!
Tri-Klops used a sword, but it wasn't his main weapon/power - just like how Fisto carries a sword.
Blade for sure. He was one of the figures I vividly remember buying and he was one of my favorites. 2 swords and no depth perception and he's still the best swordsman. What a guy.
Blade of course!
Tri-Klops used a sword, but it wasn't his main weapon/power - just like how Fisto carries a sword.
MOTUC WISHLIST - Karg | Lady Slther | Kothos | Staghorn | Sorceress of Serpos Teela | Hunga the Harpy | Kittrina | Dylamug | Hawke | Angast | Zilora | Artilla | Quakke | Robo-Woman | Great Black Wizard | Enchantress | Red Beast | Delora | DC He-Man | DC Skeletor | Lodar | Fang-Or | Mini Comics Tri-Klops | Filmation Jitsu, Frosta & Kobra Khan | Hans Hammer Holder | Granita |
Based on who all I have seen in action in all the series, comics, and movie I'd have to say Skeletor (MYP) and Tri-Klops (MYP). Blade did not impress me in the movie.
View my collection and customs HERE!
Tri-Klops, hands down...
Check out my MASTERS OF THE UNIVERSE ART & More at my ART BLOG!!!
JafariStew's ART BLOG!! UPDATED 3.31.17!!
Jafaristew...the guy that coined the term "200X."
ten characters more required to reply to this thread... wow.
Odds, Bobs, Hammer and Tongs!
I can't believe none of us have posted a Heroic Warriors version of this thread yet!
MOTUC Most Wanted: Skeleteen, Prince Keldor, Captain Randor, The Great Black Wizard, Lady Slither, Spirit, Tuvar, Baddra, Cursed Marzo, and for the love of Grayskull, ODIPHUS!!!
Instagram: Just_Thommy
MOTUC Most Wanted: Kayo, Vizar, Hoove, Lizorr
UK MOTU Fan Club Members - Add You Member Name to this Thread
Trade my Red Stone He-Ro Staff for a Green one
"Wheresoever on earth he dwells, man is prey to two weaknesses: the need to pray and the need to love."-Marquis de Sade
"It is not by reasoning or by our understanding that we have received our religion; it is by external authority and command."-Michel De Montaigne
Heretical Vintage Purist and Non-Fan Extraordinaire!
1. Zodak -is an immortal martial artist. Though we've never seen him use a sword, I'm sure he's best on Eternia in all forms of combat and with all weapons. After he gave his staff to Evil-Lyn and Rattlor (which we're to believe is his best/most powerful weapon) all he did was walk over to his weapons rack and choose another (mace). With this secondary and far less powerful weapon (I doubt all his weapons can rip/seal dimensional fabric) he fought the "most powerful man in the universe" (who of course always wields the most powerful weapon) to a stand still- all the while regarding him as little more than a nuisance.
2. King Grayskull- It's still a mystery where his great power originates being that most modern Eternian Power originates from him. Where ever it comes from, I'm sure it was mostly the reasoning he could fight off droves of Snake Men. I imagine KG used his battle axebefore He-Ro fashioned him his sword. But then he used the sword(s) to ultimately defeat the armies of Hordesman thus putting him high on this list.
3. Green Goddess- much like Zodak, she's an immortal and her only job is combat training. Though her storyline is undeveloped and very much feels shoe-horned, we still have to factor her in this contest.
4. Dekker -if Randor, Duncan, and Malcolm are the best swordsman we can think of for Heroic Warriors, it's safe to say the old master that taught them all is that much better. And much like Zodak- who we've never seen use a sword yet still adept with whatever- Dekker helped fight off a group of Eternia's strongest monsters with a piece of driftwood. I wonder if driftwood mastery is still taught at the royal academy...?
5. Fisto -The man uses two very distinct swords (a regular one and very large one) that I'm sure he was proficient with before his handicap turned superpower.
6. He-Man -Though I've never considered He-Man a master swordsman either, he did beat Blade in the movie. Blade would've really been in trouble if there were any big rocks around. Lol.
7. Teela -she was always practicing her sword or combo. Still too young to be considered high on the list or best in Eternia. She's still too young and brash to be considered for other titles she holds either IMO.
The few not factored in this contest is due to faction (She-Ra- TGR) or lack of sufficient evidence (Sir Laser-Lot, He-Ro) etc.
I forgot about Skeletor, he was a cool swordsman in MYP.
Even though Tri-Klops carries a sword, I have never seen him use it in any media
MOTUC WISHLIST - Karg | Lady Slther | Kothos | Staghorn | Sorceress of Serpos Teela | Hunga the Harpy | Kittrina | Dylamug | Hawke | Angast | Zilora | Artilla | Quakke | Robo-Woman | Great Black Wizard | Enchantress | Red Beast | Delora | DC He-Man | DC Skeletor | Lodar | Fang-Or | Mini Comics Tri-Klops | Filmation Jitsu, Frosta & Kobra Khan | Hans Hammer Holder | Granita |
See also:
MOTU Icons of Evil Tri-Klops.jpg
He's pretty BA.
Tri-Klops used his sword against He-Man in the MYP Council of Evil episodes. I think that most people who vote for Tri-Klops base their opinion of him as the master swordsman on either the early mini-comics were he used the sword as a kind of Ronin bounty hunter, the 200x comics or those episodes in MYP were he went head-to-head with He-Man. At least, that's what comes to mind for me.
I was always of the opinion as a kind of sword and sorcery society they have in MOTU just about every warrior character, male or female, would be proficient in combat with a sword. Which is the best of the bad guys is tough. To me it's a toss up between Keldor/Skeletor, Tri-Klops and Count Marzo. My reasoning is simple; in MYP those were the only evil characters who faced off against He-Man with a sword telling me not only are they master swordsmen but that they have the confidence in their training and skill to take on the most powerful man in the universe one-one-one, or in the case of Skeletor two-on-one, with He-Man's weapon of choice. It would be like arm-wrestling Superman; you'd have to be strong to compete.
A journey that began long ago in a place neither near nor far
A story of heroes, passed down through the ages
Until now.
This is our story. These are our heroes
Let's not forget Roboto, who is seen wielding a sword in that vintage poster!
And yes, there are fewer swordsman among the heroes, but it only takes two for a debate.
MOTUC Most Wanted: Skeleteen, Prince Keldor, Captain Randor, The Great Black Wizard, Lady Slither, Spirit, Tuvar, Baddra, Cursed Marzo, and for the love of Grayskull, ODIPHUS!!!
Instagram: Just_Thommy
Most wanted: Starburst She-ra, Lodar, Vintage Toy Catra, Lady Slither, and the Great Black Wizard!
Love this thread but I can't and won't say who could take who in a battle of swordsmanship. as in my opinion all three main candidates are very closely matched and on the right day anyone of them could take the other. Due to his recent bio I'd favour Jitsu , I think him and Tri-Klops would have a camaraderie between them.


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Stew meat adds a bit more taste and texture to chili than ground beef. You don't need to brown and drain it so prep time is reduced.
Serve leftovers tucked into taco shells or tortillas for quick-to-fix tacos.
For Tex-Mex toppers, try shredded coconut, rice and pecans. In the Texas panhandle and into New Mexico, this is called a "pile-on."
Ready-to-heat meals are always welcome in homes with new babies. Pack chili in one- or two-serving-size containers and freeze. Label the containers with the contents and add whimsical baby stickers before packing in a pretty gift bag.
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The discovery of tapes of Graham Chapman reading from his 1980 A Liar's Autobiography has made it possible for the expired Monty Python member to star, posthumously, in his own biopic. A trio of directors (Ben Timlett, Jeff Simpson, and Bill Jones, son of Terry), working with 14 animation studios, crafted a movie that, like the book, is anarchic, witty, frank, raunchy, and dark. In it, Chapman 'fesses up about his many excesses, which include swilling gin, sexual promiscuity (mostly with men, but also with women), and smoking a pipe (he died at 48 from throat cancer). Neither a Python movie nor strictly a comedy, this enter-taining film invites the question: was Chapman a warrior against convention and hypocrisy, or simply a hedonist who did whatever he could get away with?
Biographical episodes, done in various animation styles, feature newly recorded bits by Terry Jones, Michael Palin, and John Cleese edited into Chapman's voice-over. In a comic highlight, they play their young selves, depicted as monkeys, bickering over titles for their TV show. Jones and Palin also play Chapman's dotty Mum and gruff Dad. The medium allows for trippy fantasy (Chapman cruises in a cockmobile) and expressionism (an ink-on-glass animation of Chapman suffering through self-imposed detox from alcohol is intercut with the Python sketch in which he wrestles himself). It's an affectionate, imaginative tribute to the artist's idiosyncratic take on life.
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Deeplinks Blog posts about Hollywood v. DVD
Great gadgets for your music collection are all over CES: servers that stream to devices throughout your house, slick portable players and music cell phones, place-shifting software that lets you -- and your friends -- hear your collection from any computer, and much more. But if you want to do more with your DVD collection, you can basically forget about it.
"Apparently, Hollywood believes that you should have to re-purchase all your DVD movies a second time if you want to watch them on your iPod." That's what I said last week, commenting on the Paramount v. Load-N-Go lawsuit, in which Hollywood studios claimed that it is illegal to rip a DVD to put on a personal video player (PVP), even if you own the DVD.
Well, this week the other shoe dropped. According to an article in the New York Times:
Customers who buy the physical DVD of Warner Brothers' "Superman Returns" in a Wal-Mart store will have the option of downloading a digital copy of the film to their portable devices for $1.97, personal computer for $2.97, or both for $3.97.
The MPAA studios are at it again, snatching away our fair use rights, so they can sell them back to us for an "additional fee."
In a lawsuit filed in federal court in New York, Paramount Pictures v. Load 'N Go Video, the MPAA member companies have sued a small business for loading DVDs onto personal media players (e.g., iPod Video) on behalf of customers.
According to the suit, Load 'N Go sells both DVDs and iPods and loads the former onto the latter for customers who purchase both. The company then sends the iPod and the original DVDs to the customer. So the customer has purchased every DVD, and Load 'N Go just saves them the trouble of ripping the DVD. The movie studios' suit claims that this is illegal, because ripping a DVD (i.e., decrypting it and making a copy) is illegal under the DMCA. The suit also claims that this constitutes copyright infringement.
December 1 is the last day to submit proposals (by 5p EST) to the Copyright Office seeking a 3-year DMCA exemption for noninfringing activities that are otherwise squelched by "digital rights management" (DRM) restrictions.
As we mentioned back in October, Congress has instructed the U.S. Copyright Office to consider every three years whether we need temporary exemptions to the DMCA's blanket ban on circumventing "technological protection measures" (aka DRM) used to lock up copyrighted works.
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Origin of nascent lineages and the mechanisms used to prime second-strand DNA synthesis in the R1 and R2 retrotransposons of Drosophila
- Deborah E Stage1 and
- Thomas H Eickbush1Email author
DOI: 10.1186/gb-2009-10-5-r49
© Stage and Eickbush; licensee BioMed Central Ltd. 2009
Received: 27 January 2009
Accepted: 5 May 2009
Published: 5 May 2009
Most arthropods contain R1 and R2 retrotransposons that specifically insert into the 28S rRNA genes. Here, the sequencing reads from 12 Drosophila genomes have been used to address two questions concerning these elements. First, to what extent is the evolution of these elements subject to the concerted evolution process that is responsible for sequence homogeneity among the different copies of rRNA genes? Second, how precise are the target DNA cleavages and priming of DNA synthesis used by these elements?
Most copies of R1 and R2 in each species were found to exhibit less than 0.2% sequence divergence. However, in many species evidence was obtained for the formation of distinct sublineages of elements, particularly in the case of R1. Analysis of the hundreds of R1 and R2 junctions with the 28S gene revealed that cleavage of the first DNA strand was precise both in location and the priming of reverse transcription. Cleavage of the second DNA strand was less precise within a species, differed between species, and gave rise to variable priming mechanisms for second strand synthesis.
These findings suggest that the high sequence identity amongst R1 and R2 copies is because all copies are relatively new. However, each active element generates its own independent lineage that can eventually populate the locus. Independent lineages occur more often with R1, possibly because these elements contain their own promoter. Finally, both R1 and R2 use imprecise, rapidly evolving mechanisms to cleave the second strand and prime second strand synthesis.
Transposable elements (TEs) are ubiquitous components and extensive manipulators of eukaryotic genomes. Because TEs constitute a significant mutation source and their remnants often comprise the majority of genomes, they are usually regarded as genomic parasites that are occasionally co-opted for host benefits [1, 2]. While tracing the evolution of any genome should include a description of the natural history of its transposable elements, the diversity of TEs and their histories are so extensive that even with the advent of genome sequencing and assembly it remains challenging to follow the interplay between TEs and their host.
Evolution of the rDNA locus is known to be dominated by concerted evolution, a recombinational process involving unequal crossovers and gene conversions that maintain near identity among repeats within a species while allowing those repeats to diverge between species [15]. Abundant evidence corroborates the extremely low sequence variation present among the many copies of the rDNA unit [16–18]. Sequence variants present at the lowest frequencies are equally distributed between the coding and non-coding regions of the unit. In contrast, the rare variants present at higher frequencies are greatly enriched in non-coding regions, indicating that selective pressures guide the extent of standing variation within the locus [18].
In arthropods from a few percent to over 50% of the rDNA units are inserted by R1 or R2 elements [19], and those units are thus prevented from producing functional 28S rRNA [20]. Within a species these many copies of R1 and R2 elements also exhibit low levels of sequence variation [21]. Surprisingly, divergent lineages of R1 or R2 are frequently found in a species, which cannot be explained by horizontal transfers between species [22]. This suggests that divergent lineages of elements must be able to form within a species.
The rDNA locus is not assembled as part of genome projects because of the highly repetitive nature of the rDNA locus. Thus, in this report we used the original sequencing reads generated from the 12 Drosophila genomes project [23] to address specific questions concerning the evolution and mechanism of integration of R1 and R2 elements. Can different lineages of R1 and R2 arise within a species despite concerted evolution maintaining sequence homogeneity among the rRNA genes? What is the location of second-strand DNA cleavage? How is this site used to prime second-strand synthesis in the retrotransposition reaction?
Results and discussion
We also searched in all species for R2 copies that might be present outside the rDNA locus. We found no extra-rDNA R2 copies in D. melanogaster, as previously reported [27], or in D. ananassae or D. persimilis. D. pseudoobscura, D. sechellia, D. simulans, D. willistoni, and D. yakuba each had R2 copies not inserted in a 28S gene. These copies were frequently incomplete and all contained sequences that were from 1% to 2% divergent from those R2 copies within the rDNA locus. Thus, these non-rDNA copies of R2 could not have given rise to the current populations of R2 insertions in the rDNA locus. Finally, in D. simulans a fusion of the 5' end of an R1 element with the 3' end of an R2 element was identified as a tandem array outside the rDNA locus.
Complete R1 elements were assembled in all 12 sequenced genomes (Figure 2b; Additional data files 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21). The coding capacity of all R1 ORFs was consistent with previously characterized R1 elements [24]. A test of selection by dN/dS analysis indicated that the assembled R1 elements had undergone purifying selection (R1A, mean dN/dS = 0.30 with standard deviation of 0.376; R1B, mean dN/dS = 0.27 with standard deviation of 0.348). Previous analyses of R1 elements in Drosophila have suggested there are two distinct lineages of elements, A and B, that separated well before the origin of this genus and are differentially retained in the various species lineages [28]. Eleven of the sequenced Drosophila species contained a single R1 family of either the R1A or R1B lineage, while D. ananassae contained both lineages (Figure 2b). The only consistent difference in structure between the two lineages was that the two ORFs in the R1A lineage overlapped by 7 bp with a corresponding frame shift of -2, while the ORFs in the R1B lineage had a frameshift of -1 and overlapped from 14 bp in D. ananassae to 59 bp in D. grimshawi. As will be described below, in most species multiple examples were also identified of R1 insertions in non-28S gene locations.
R1 and R2 intraspecies sequence variation
Variation in the 5' and 3' ends of R1 and R2 elements
Major copy type: mean divergence* (maximum)
Atypical sequences: number (divergence)
5' end
3' end
Variant copies†
Variant 5' ends‡
R1 elements
D. simulans R1A
0.000 (0.000)
0.002 (0.003)
4 (0.01-0.03)
D. sechellia R1A
<0.001 (0.003)
<0.001 (0.002)
D. melanogaster R1A
0.001 (0.008)
0.003 (0.015)
2 (0.07)
D. yakuba R1A
0.001 (0.005)
0.000 (0.000)
1 (0.02)
D. erecta R1A
0.002 (0.005)
0.001 (0.007)
D. ananassae R1A
0.013 (0.043)
<0.001 (0.003)
1 (0.08-0.11)
D. ananassae R1B
0.000 (0.000)
0.002 (0.005)
3 (0.15-0.22)
D. pseudoobscura R1A
0.000 (0.000)
0.002 (0.010)
1 (0.04)
D. persimilis R1A
0.000 (0.000)
1 (0.02)
D. willistoni R1A
0.002 (0.005)
0.001 (0.003)
D. mojavensis R1A
0.000 (0.000)
0.000 (0.000)
D. virilis R1B
0.001 (0.008)
<0.001 (0.003)
6 (0.01-0.05)
D. grimshawi R1B1
0.000 (0.000)
0.001 (0.005)
D. grimshawi R1B2
0.000 (0.000)
0.000 (0.000)
R2 elements
D. simulans R2
0.001 (0.008)
0.000 (0.000)
D. sechellia R2
0.000 (0.000)
<0.001 (0.003)
D. melanogaster R2
0.002 (0.005)
0.001 (0.015)
6 (0.02-0.05)
D. yakuba R2
0.002 (0.013)
0.006 (0.018)
D. ananassae R2
0.004 (0.008)
0.001 (0.005)
D. persimilis R2
D. pseudoobscura R2
0.005 (0.010)
D. willistoni R2 sub1
0.001 (0.003)
0.002 (0.008)
D. willistoni R2 sub2§
0.003 (0.006)
0.015 (0.028)
Nascent subfamilies of R1 and R2
In addition to the many highly uniform copies of R1 and R2, five Drosophila species had one or more copies of R1 or R2 with nucleotide divergence of 1% to 7% from the consensus, clearly outside the range of divergences seen for the remaining R1 and R2 copies. The number and level of divergence of these atypical copies are listed in Table 1. Among these copies two had premature stop codons, indicating that they were inactive, while the remaining copies appeared to have intact ORFs. Because most divergent copies were not inserted into 28S genes that were also divergent, these R1 and R2 copies could represent distinct retrotranspositionally competent lineages of elements. However, the number of trace reads suggested these divergent R1s and R2s were at single copy levels, and thus it was likely that they had not recently been active.
Stronger evidence for the formation of nascent subfamilies was found in the examples of distinct 5' ends for the R1 elements of three species ('Variant 5' ends' column in Table 1). In D. simulans there were five distinct sequence classes of R1 5' ends, with each class representing from 11% to 26% of the total number of copies. There was no nucleotide divergence within each class, while divergence between classes ranged from 1% to 3%. In the case of D. pseudoobscura there were two distinct 5' ends. One-third of the R1 copies had 5' ends with over 4% nucleotide divergence from the remaining two-thirds of the R1 copies. Finally, R1 elements in D. ananassae showed the greatest tendency to diverge into subclasses with distinct 5' ends. The R1A elements could be separated into two classes that differed by 10% in nucleotide sequence, while the R1B elements could be separated into four classes that differed by 15% to 22% in sequence.
The separate lineages of the R1 5' ends observed in these three species were not apparent at the 3' ends of the R1 elements (that is, there was one class of 3' ends with mean levels of divergence less than 0.2%). Previous authors have suggested that new sublineages of transposable elements can arise within the same species by the acquisition of new promoter sequences [29, 30]. Thus, one possibility is that the different 5' ends of R1 elements in a species correspond to rapidly evolving promoter sequences. R1 elements have been suggested to contain their own promoters because in some insect lineages R1 inserts in the opposite orientation in the 28S gene, or even outside the rDNA locus [9, 31]. R2 elements on the other hand appear to be co-transcribed with the 28S gene and thus do not have their own promoter [32, 33]. No evidence of divergent 5' ends was found for the R2 elements of any Drosophila species.
A second example of the presence of subfamilies within a species was found for the R2 elements in D. willistoni. In this case one subfamily, R2.1, was highly abundant while the R2.2 subfamily (Additional data file 23) was present in only a few copies. As shown in Figure 4b the subfamilies have diverged by 4.7% in their 5' UTRs and 3.6% in their 3' UTRs, similar to the divergence between the R2 elements of D. melanogaster and D. simulans. The amino acid divergence of the ORF from the two subfamilies (2.6%) was also similar to the divergence between D. simulans and D. melanogaster (2.4%), suggesting the divergence time between the D. willistoni subfamilies is similar to the time of divergence of D. melanogaster and D. simulans.
Unequal crossover events occurring within R1 (or R2) elements would homogenize their sequences, thus preventing the separation of two distinct lineages. Because the nucleotide divergence for most of the region encoding ORF2 of the R1B.1 and R1B.2 elements in D. grimshawi was less than 1%, and thus could still undergo recombination, we looked for evidence of such events. Blast searches were conducted using a query from the end of each subfamily. We then examined the sequence trace from the other end of each approximately 3.5 kb plasmid to determine whether it contained sequence from the same or opposite subfamily. Of 115 informative plasmid ends examined, only one pair indicated recombination between the subfamilies. This paucity of recombination can explain how these nascent subfamilies are able to avoid concerted evolution and remain independent lineages.
Mechanism of R2 retrotransposition
Analysis of R2 junctions
In vitro studies with the B. mori R2 endonuclease suggested the location of top-strand cleavage occurred 2 bp upstream of the bottom-strand site (Figure 5c) [10]. Previous analyses of a few R2 5' junctions from each of several Drosophila species, as well as other insect species, were interpreted in a manner that was consistent with such a cleavage [36, 37]. However, there is significant variation at the 5' junctions of R2 elements, and the comprehensive analysis of this variation made possible using the genomic sequences suggested a reevaluation of this second-strand cleavage location was needed.
Similar to most non-LTR retrotransposons, R2 insertions can be full-length or contain truncations of their 5' ends. The 5' truncations have been suggested to be due to the failure of the reverse transcriptase to copy the entire RNA template, degradation of the RNA template, or the initiation of second-strand synthesis before reverse transcription is completed [5]. Figure 5b shows representative examples of full-length and 5'-truncated R2 elements. All full-length examples are from D. melanogaster but are representative of the R2 elements observed in all Drosophila species. Almost two-thirds of the full-length insertions have 5' junctions that include the 28S sequence to the position across from the site of bottom-strand cleavage, position '0' (examples a, d and e), with the remaining third containing variable deletions of the upstream 28S sequence (examples b and c). Many junctions contain additional bases at the junction. In some cases the additional bases represented duplications of the 28S gene (example e), while for other junctions the origin of the additional bases could not be identified, here called non-templated bases (example d). In the case of the 5' truncated elements most junctions contain from one to five bases at the precise 28S/R2 junction that may be assigned to either 28S or R2 (examples of these microhomologies are indicated by the shaded bases in Figure 5). R2 5' truncated junctions can also be associated with deletions of upstream 28S sequences (examples g and h), and non-templated additions (example i).
R2 retrotransposition model
This analysis of the 5' junctions of R2 insertions supports the following additions to the TPRT model for R2 retrotransposition. First, the most frequently used top-strand cleavage site in Drosophila is directly opposite the bottom-strand site, rather than 2 bp upstream (position -2) as suggested from the in vitro studies with the R2 endonuclease from B. mori [10]. Cleavage opposite the bottom-strand site readily explains junctions such as examples d, e and i in Figure 5b, junctions difficult to explain if top-strand cleavage was at position -2. Second, we suggest that full-length R2 RNA transcripts in Drosophila contain G residues at their 5' end. All integrated full-length R2 copies in D. melanogaster begin with four G residues (examples a to e in Figure 5b), and similar analysis of the other Drosophila species indicated that the full-length R2 elements in these species contained at least two terminal G residues (data not shown). No conservation of R2 5' end sequences is found beyond these two Gs.
To explain the most frequently observed R2 full length junctions (example a in Figure 5b), we suggest that two terminal C residues on the cDNA strand made from the R2 transcript anneal to the cleaved target site (see Figure 5c for a diagram). A tendency to anneal a few nucleotides of the cDNA strand to the top strand of the target DNA before initiating second-strand DNA synthesis would explain the frequent 'microhomologies' between internal R2 sequences and upstream 28S sequences that are found associated with 5' truncated R2 insertions. The non-templated nucleotides found at the 5' junctions of full-length and truncated copies of R2 are suggested to result when the annealing of microhomologies does not occur. In vitro studies have shown that the R2 polymerase adds non-templated nucleotides before initiating from a primer that is not annealed to the template [38], as well as when it 'runs-off' the end of a template [39]. These non-templated nucleotides can be of any sequence and thus could also lead to microhomologies used to initiate polymerization of the top DNA strand. Microhomologies between these non-templated nucleotides and the 28S gene would go undetected by our analysis. Finally, the R2 junctions with deletions of the 28S gene, as well as the few with duplications of the 28S gene, could represent top-strand cleavages outside the preferred site.
Mechanism of R1 retrotransposition
R1 junction sequences
Based on their 5' junctions the R1 elements could be divided into two groups. For R1B elements and the R1A elements outside the melanogaster species group, the upstream 28S gene sequences typically extended to a position 14 bp downstream of the bottom-strand site (+14), and thus a 14 bp target site duplication (TSD) flanks the R1 insertions (Figure 7, left side). Because similar length TSDs flank the R1 elements in most other arthropods [40, 41], this group is called the 'ancestral type' R1 insertions. All sequence variation associated with the 5' junctions of both full-length and 5' truncated elements of the ancestral type were located at the end of the TSD (bracketed region in Figure 7). As with the R2 elements, these 5' junctions could be classified as precise, containing microhomologies, or containing non-templated nucleotides. Most full-length ancestral type R1 insertions were precise, with the remainder containing non-templated nucleotides. The 5' truncated ancestral R1s are more broadly distributed between precise, non-templated and microhomology junctions.
In contrast to the ancestral type R1s, many copies of the R1A elements in the melanogaster species group (D. ananassae, D. erecta, D. melanogaster, D. sechellia, D. simulans, and D. yakuba) contained upstream 28S gene sequences that extended only to position -9. The relative proportion varied from species to species, but altogether 75% of the full-length 'melanogaster-type' R1 insertions contained this 9 bp deletion. No 5' sequence variation was associated with these insertions. The remaining full-length insertions contained variable-length TSDs up to 17 bp in length and an unusual duplication of 28S sequences that we called insertion site rearrangements (ISRs). These duplications of the 28S sequence extended for 16 to 27 nucleotides upstream of position -9. Microhomologies and non-templated nucleotides were common at these junctions and were always located between the TSD and the ISR (bracketed region). The 5' truncated insertions of the melanogaster-type R1s also contained precise, non-templated and microhomology junctions.
Tandem R1 arrays
Ratio of R1 elements in the canonical 28S site, tandem arrays and non-28S locations
Fraction of rDNA units
D. simulans
D. sechellia
D. melanogaster
D. yakuba
D. erecta
D. ananassae R1A
D. ananassae R1B
D. pseudoobscura
D. persimilis
D. willistoni
D. mojavensis
D. virilis
D. grimshawi
R1 insertions into non-28S locations
In most Drosophila species copies of R1 were also identified that had inserted into sequences outside the 28S gene (Table 2). Frequent R1 insertions outside the 28S gene have also been reported in B. mori [43]. The Drosophila species with the most abundant examples of non-28S R1 insertions were D. ananassae and D. pseudoobscura, the two species with the highest levels of tandem duplications. This may suggest that R1 insertions in these species are either less specific or retrotranspose more often. We identified 118 unique examples of non-28S gene R1 insertions that had intact 3' junctions, suggesting that they represented authentic retrotransposition events, not segments of R1 sequence that had been displaced by recombination to locations outside the rDNA locus. The insertion sites for these copies frequently corresponded to repeated DNA sequences, probably in the pericentromeric or telomeric regions of the genome. An exception was in D. ananassae, where a 22 bp region of the 28S gene corresponding to the R1 28S insertion site was found in the IGS region of the rDNA unit. One R1A element and seven R1B elements were found in rDNA units containing this unusual insertion of 28S sequences within the IGS.
The 5' junctions of all melanogaster-type full length R1s outside the rDNA array contained upstream 28S gene sequences ending at position -9 and extending upstream for 7 to 28 bp (Figure 9, examples 5 to 10). The 5' ends of the ancestral type R1 insertions showed no evidence for the insertion of flanking 28S rRNA sequence (examples 1 to 4 and 11). The 3' junctions of both the ancestral and melanogaster-type R1s were similar, with most beginning at the precise 3' junction of the R1. However, some insertions showed the incorporation of up to 14 nucleotides of downstream 28S sequences (example 13) or the presence of non-templated nucleotides (example 12).
R1 retotransposition model
The analysis of the R1 5' junctions with the 28S genes, as well as the insertions into non-28S gene locations, enables us to propose several steps involved in the R1 retrotransposition reaction. These suggestions are based on the standard TPRT mechanism of R2 and other non-LTR retrotransposons and the ability of their polymerases to add non-templated nucleotides at the ends of synthesized DNA strands. First, because no variation was detected within a species or between species for the location of the bottom-strand cleavage used to prime reverse transcription, like R2, this is the most conserved step in the retrotransposition reaction of R1. The analysis of R1 insertions outside the 28S gene site revealed that downstream 28S gene sequences are sometimes also inserted with the R1 element. In addition, many of these non-28S gene target sites contain bases corresponding to the 28S gene (examples 1 to 6 in Figure 9). Together these data suggest that the 3' end of the R1 transcript used for retrotransposition often contains flanking 28S sequences. These flanking 28S sequences may anneal to the bottom strand of the target site and thus account for why there is little sequence variation associated with the 3' junctions of R1.
R1A lineage elements in the melanogaster species group appear to have evolved changes in the TPRT reaction. In these R1s, top-strand cleavage frequently occurs 9 bp upstream of bottom-strand cleavage, resulting in a 9 bp target site deletion. Priming of second-strand synthesis from this site is highly precise, that is, non-templated sequences were never observed, suggesting 28S gene sequences are included at the 5' end of the RNA transcripts used for retrotransposition. This flanking 28S sequence allows the reverse transcribed strand to anneal to the target site (Figure 10, middle). Consistent with this suggestion, from 7 to 28 bp of flanking 28S sequences ending at position -9 are associated with the non-28S gene insertions of the melanogaster-type R1. The R1A elements in the melanogaster group also appear able to cleave the top DNA strand in a less specific manner around position +14 (Figure 8b). Such cleavages result in ISR junctions in which a typical TSD is present followed by 16 to 27 bp of the 28S gene ending at position -9 (Figure 10, right side). Considerable variation is detected in these junctions between the TSD and the duplicated upstream 28S sequences, presumably because this downstream cleavage eliminates the ability of cDNA sequences to anneal to the top strand in the initiation of second-strand synthesis. An intriguing aspect of these melanogaster-type R1 elements is that all 5' truncated insertions have TSDs (Figure 8), suggesting they can only arise from downstream cleavage of the top strand. One possibility is that the default cleavage site for the R1 endonuclease is downstream of the insertion site, but the 5' end of the full-length R1 transcripts acts as a signal directing the cleavage site to the position at -9.
Origin of nascent lineages
The availability of whole genome shotgun sequences has enabled us to evaluate the level of sequence variation of the R1 and R2 elements in 12 species of Drosophila. The level of nucleotide divergence for most copies was typically less than 0.2%, suggesting either the elements are subject to the same concerted evolution mechanisms that enable the rRNA genes themselves to remain nearly identical, or that the R1 and R2 elements are gained and lost rapidly from the locus (that is, all copies are recent insertions). All previous analysis suggested the latter explanation. Analyses of the 5' truncated copies of R1 and R2 in several species have suggested that these elements do turnover rapidly. Different animals from the same population were found to have different collections of 5' truncated copies [44, 45] and most 5' truncated elements within an animal had not undergone duplication by recombination [46, 47]. Thus, the individual copies of R1 and R2 do not appear to remain in the rDNA locus for long enough periods to be substantially influenced by the recombinations leading to the concerted evolution of the locus.
One puzzling aspect concerning the evolution of R1 and R2 was the presence of multiple families in some species, yet no evidence for the origin of these lineages by horizontal transfer [28, 48]. The rapid turnover of individual R1 and R2 elements suggests that each active copy can generate its own lineage, which over time should accumulate sequence variation. Thus, while separate lineages of R1 and R2 should be able to arise, the question remained as to whether they could be maintained within a species. In this report we have for the first time detected these nascent lineages of R1 and R2 elements within a species. Two distinct subfamilies of R1B elements were detected in D. grimshawi and two distinct subfamilies of R2 elements in D. willistoni (Figure 4). In addition to these distinct lineages, other species contained individual copies of R1 or R2 that had from 1% to 7% nucleotide divergence from the majority of elements in the species. Many of these divergent copies had intact ORFs, and thus were potentially active. Another frequent finding was the presence of distinct 5' UTRs for the R1 elements (Table 1). This was most widespread in D. ananassae where the R1A elements could be divided into two groups that diverged 10% in their 5' UTR sequences, while the R1B elements could be divided into four groups with 5' UTRs that diverged from 15% to 22%. We suggest this accelerated divergence of the 5' UTRs represents the evolution of new promoter sequences driving the transcription of the elements. Once a new promoter is formed, copies containing this new promoter may differ in their expression, thus giving rise to new lineages of elements.
Similar examples of the rapid evolution of the 5' UTRs of R2s were not detected. We have previously suggested that R2 elements do not contain their own promoter but are co-transcribed with the 28S gene [32, 33]. This co-transcription may make it more difficult for independent R2 lineages to evolve. A single lineage of R2 elements has been found in the Drosophila genus [25], while four distinct lineages of Drosophila R1 elements exist [28]. It is also possible that the more frequent loss of R2 from a Drosophila species (3 out of 12 species) compared to R1 (no losses among the 12 species) could also be the result of R2's reliance upon co-transcription with the rDNA units in which they reside.
Second-strand DNA synthesis
Many of the initial steps involved in the cleavage of the target site bottom strand and its use to prime reverse transcription have been characterized using purified R2 protein in vitro [10, 38, 49]. The recent discoveries that sequences near the 5' end of the RNA transcript regulate top-strand cleavage [11], and that the R2 polymerase can efficiently displace an RNA strand while using a DNA strand as template [50], suggest that the R2 protein also synthesizes the second DNA strand. However, many questions remain concerning whether and how top-strand cleavage of the target DNA is used to prime second-strand synthesis.
Because of an unusual template jumping ability of the R2 polymerase [39], we previously suggested that during reverse transcription the R2 polymerase jumps from the R2 transcript onto the upstream DNA target sequences [5, 36, 37]. The hundreds of R2 5' junctions analyzed here suggest a different model. In Drosophila we propose that the 5' ends of the R2 RNA transcripts contain terminal G residues that, after reverse transcription and top-strand cleavage, enable the terminal C residues to anneal to the G residues of the top DNA strand after cleavage (Figure 5c). This model would explain the most common full-length R2 insertions in all Drosophila species. Similarly, microhomologies between internal R2 sequences and the upstream 28S gene sequences can explain the priming of second-strand DNA synthesis in many 5' truncated R2 insertions. When annealing of microhomologies does not occur, non-templated residues can be observed that were either added during first-strand synthesis when the R2 polymerase runs off an RNA template [39] or during second-strand synthesis before the R2 polymerase engages the cDNA [38].
The only aspect of this integration model that does not agree with previous biochemical studies is the location of the top-strand cleavage site. The experiments using the B. mori R2 protein suggested this cleavage was two nucleotides upstream of the bottom-strand site [10] while our analysis of Drosophila R2 junctions suggest it is opposite the bottom-strand site (Figure 5c). We suggest this represents an evolved difference between the R2 elements in these divergent insects. We have analyzed the 5' junctions of over 40 B. mori R2 insertions and found their structure is consistent with the location of top-strand cleavage determined with the purified protein [37]. We suggest that cleavage of the top strand by the R2 endonuclease is not rigidly determined, and thus its location can vary.
Much less was previously known about the R1 retrotransposition mechanism. In vitro studies of the R1 endonuclease, again from B. mori, revealed a bottom-strand cleavage site in a position consistent with its use to prime reverse transcription [12, 13]. All Drosophila R1 3' junctions, as well as 3' junctions in other insect species [19], are consistent with this cleavage site. As in the case of R2, the R1 5' junctions suggest cleavage of the top strand is less precise and subject to evolutionary changes. For many Drosophila R1 insertions top-strand cleavage was proposed to be at a site 14 bp downstream of the bottom strand, again consistent with the biochemical studies. Use of this site to prime second-strand synthesis results in a 14 bp TSD. Minor changes in this cleavage location were found in D. ananassae, where the R1B insertions have 11 or 13 bp TSDs, and in D. willistoni, where some ancestral type R1A insertions have an 8 bp TSD. Annealing of cDNA sequences to the upstream target site presumably does not occur as significant nucleotide variation is observed at the junctions.
A radical change in top-strand cleavage site preference is proposed for the R1A elements of the melanogaster species group. In these species, many R1 insertions result in a 9 bp target site deletion, suggesting cleavage frequently occurs 9 bp upstream of the bottom-strand site (Figure 10). Associated with this remarkable change in cleavage site preference also appears to be a change in the nature of the R1 RNA transcript used for retrotransposition. Based on the structure of the ISR full-length insertions and of non-28S insertions, these melanogaster-type R1 transcripts contain from 6 to 27 nucleotides of 28S sequence ending 9 bp upstream of the integration site. These upstream 28S sequences on full length R1 transcripts appear to anneal to the target site, giving rise to highly precise 5' junctions. Interestingly, these melanogaster-type R1s retain the ability to cleave downstream of the integration site, resulting in the ISRs seen in some full-length insertions and the TSDs observed for all 5' truncated insertions. Because annealing of the upstream 28S sequences is not possible with these downstream cleavage sites, significant variation is observed at these junctions. We searched for evidence of these additional 28S sequence tags among available expressed sequence tag sequences from D. melanogaster. However, all R1 sequences contained extensive upstream 28S sequences or were transcripts of R1 tandem arrays (data not shown).
Thus, the integration mechanisms used by the R1 and R2 elements are quite similar. Cleavage of the bottom strand and its use to prime first-strand DNA synthesis is rigidly determined, and no variation was observed between species. Cleavage of the top strand and its use to prime second-strand synthesis is flexible, which results in different junctions both within and between species. R1 and R2 are highly successful in the 28S niche. Whether change in the location of top-strand cleavage occurs simply because it is neutral or because it increases insertion efficiency is unclear. Nevertheless, the ability of R1 and R2 to explore the top-strand cleavage site suggests that variations of the retrotransposition mechanism can evolve among arthropods that could affect both the elements and the rDNA loci in which they reside.
Materials and methods
Species and databases
Original sequencing reads from the whole genome shotgun sequencing projects were accessed by Blast search (version 2.2.17) in the trace archives at NCBI [51]. The 12 Drosophila species and their sequencing fold coverage were: D. ananassae (9-fold), D. erecta (10-fold), D. grimshawi (8-fold), D. melanogaster (12-fold), D. mojavensis (8-fold), D. persimilis (4-fold), D. pseudoobscura (9-fold), D. sechellia (5-fold), D. simulans (4-fold; white 501 strain only), D. virilis (8-fold), D. willistoni (8.4-fold) and D. yakuba (9-fold).
Assembling R1 and R2 elements
Downstream 28S sequences flanking the known R1 and R2 insertion sites were used as Blast queries using default parameters except for requesting the maximum of 20,000 target sequences. The reads were collected, trimmed upstream of the query and aligned in ClustalX [52]. Those sequences upstream of the query that were not 28S rRNA sequences were considered putative R1 or R2 elements and were used as Blast queries in the nucleotide collection database (nr/nt) and VecScreen at NCBI [51]. Some putative elements were identified as cloning vector sequence and disregarded. For the remaining majority of sequences, 5' extensions of the sequences were assembled by Blast search, followed by alignment and extraction of a consensus. Iterations were done until the 5' junction of the element with the 28S was reached. Full assemblies were not possible in a few instances because of low element copy number and low coverage of some genome sequences.
Alignments of concatenated 18S and 28S sequences (from Stage and Eickbush [18] and available at the Eickbush lab website [53]) were done in ClustalX [52] with minor manipulations done in Jalview [54]. Default parameters of phyML version 3.0 [55] as implemented through the LIRMM website [56, 57] were used to construct maximum likelihood trees and visualized using TreeDyn 198 [58]. Branch support values were the minimum score based on Shimodaira-Hasegawa-like and Chi2-based approximate likelihood-ratio tests [59].
Nucleotide variation in the 5' and 3' ends of elements and sequence divergence between species
Blast queries were 75 bp long near the 5' or 3' end of each consensus R1 or R2 sequence; default Blast parameters were used except for requesting the maximum of 20,000 target sequences and using a match/mismatch score of 1,-1. The Blast results were trimmed to include 400 bp of the element and 100 bp of flanking sequence. Retention of the flanking sequence enabled those elements present in the 28S gene to be separated from those copies inserted elsewhere in the genome. ClustalX alignment [52] of results from each Blast search was conducted and groups of like sequences were extracted and consensus sequences derived. Quality scores for reads containing differences from the main consensus were examined to verify that the variation observed was not sequencing error (scores less than 40 were considered sequencing error). The Sequence Manipulation Suite: Ident and Sim [60] aided determination of the percent identity of the variants relative to the consensus.
In theory it should be possible to make copy number estimates based on the genome sequence coverage and the number of trace reads recovered. In practice, however, random variation in fold coverage for any given sequence means only an estimate of copy number for repetitive sequences can be inferred. In addition, rDNA loci in many species of Drosophila are located on the X and Y chromosomes, resulting in different sequencing coverage. Finally, by requiring analyzed reads to cover 400 bp to enable a significant and uniform level of information to be recovered from each read, fewer reads were obtained for each analysis. Thus, estimates of element abundance were made relative to the number of rDNA units also recovered in the trace archives (that is, fraction of rDNA units inserted). Copies with small numbers of reads (approximately 2 to 12) and identical sequence variation were interpreted as single elements.
Insertions outside the 28S genes
Using the 5' and 3' ends of R1 and R2 elements as Blast queries and analyzing the flanking sequences, we found many examples of R1 elements inserted outside the usual 28S site. Identification of the non-28S sequences was attempted by comparing them to our assembled rDNA-related sequences and by Blast search of the NCBI non-redundant nucleotide database [51]. While a few target sites could be identified, the majority of sequences had not been characterized and represented repeated sequences, and thus were presumed to be heterochromatic. We then attempted to find uninserted copies of the sequences using the flanking sequences as Blast queries. In those cases where the target site was repeated, we could identify examples of uninserted target sites, and in some instances the opposite junction of the R1 insertion.
dN/dS analysis
Selection analysis was done using the HyPhy package available at Datamonkey [61]. We conducted all the available tests and report here the dN/dS results of the PARRIS test [62], which were similar in all the tests.
Additional data files
The following additional data are available with the online version of this paper. Each file contains the consensus nucleotide sequence for an R1 or R2 element: Drosophila ananassae R2 (Additional data file 1); Drosophila melanogaster R2 (Additional data file 2); Drosophila persimilis R2 (Additional data file 3); Drosophila pseudoobscura R2 (Additional data file 4); Drosophila sechellia R2 (Additional data file 5); Drosophila simulans R2 (Additional data file 6); Drosophila willistoni R2.1 (Additional data file 7); Drosophila yakuba R2 (Additional data file 8); Drosophila ananassae R1A (Additional data file 9); Drosophila ananassae R1B (Additional data file 10); Drosophila erecta R1 (Additional data file 11); Drosophila grimshawi R1.1 (Additional data file 12); Drosophila mojavensis R1 (Additional data file 13); Drosophila melanogaster R1 (Additional data file 14); Drosophila persimilis R1 (Additional data file 15); Drosophila pseudoobscura R1 (Additional data file 16); Drosophila sechellia R1 (Additional data file 17); Drosophila simulans R1 (Additional data file 18); Drosophila virilis R1 (Additional data file 19); Drosophila willistoni R1 (Additional data file 20); Drosophila yakuba R1 (Additional data file 21); Drosophila grimshawi R1.2 (Additional data file 22); Drosophila willistoni R2.2 (Additional data file 23).
insertion site rearrangement
internal transcribed spacer
long terminal repeat
open reading frame
- rDNA loci:
loci encoding the tandem array of rRNA genes
transposable element
target primed reverse transcription
target site duplication
untranslated region.
We thank Danna Eickbush for comments on the manuscript. This research was funded by National Science Foundation grant (MCB-0544071) and National Institutes of Health grant (GM42790).
Authors' Affiliations
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text:
The Estate Chardonnay grapes are also grown in an ideal location south of Yountville. There, the cooler bay-influenced temperatures and breezes create fruit of superior quality and unique character.
The worldwide reputation of Burgess wines lies in the fact that they are always in perfect harmony, fully integrated to form a "total impression". Maximum varietal character, firm oak, good body, and long, complex finish are stressed. These attributes are controlled through painstaking attention to the estate vineyards and labor intensive old world winemaking techniques; all brought together with the most modern technology. View all Burgess Cellars Wines
About Napa Valley
It's hard not to think of Napa Valley when thinking of California wines. The region is, after all, the one that brought world recognition to California wine making. The area was settled by a few choice wine families in the 1960's who bet that the wines of the area would grow and flourish. They were right. The Napa wine industry really took off in the 1980's, when vineyard lands were scooped up and vines were planted throughout the county. A number of wineries emerged, from large conglomerates to small boutiques to cult classics. Cabernet Sauvignon is definitely the grape of choice here, with many winemakers also focusing on Bordeaux Blends. Whites are usually Chardonnay and Sauvignon Blanc.
Notable FactsWithin the Napa Valley lie smaller sub-AVAs that lend even more character specifics to the wines. Furthest south is Carneros, followed by Yountville, Oakville & Rutherford. Above those two is St.-Helena and finally, just granted an AVA, Calistoga. These areas are situated on the valley floor and are known for creating rich, smooth Cabernet Sauvignon, Merlot and Chardonnay. There are a few mountain regions as well, nestled on the slopes overlooking the valley AVAs. Those include Howell Mountain, Stags Leap and Mount Veeder. Wines from the mountain regions are often more structured and firm, benefiting from more time in the bottle to evolve and soften.
About California
The most famous of the California wine regions is Napa Valley, and these wines are certainly outstanding – but it's not as broad and diverse as its larger neighbor, Sonoma County. Down south, Santa Barbara's Santa Maria Valley is well-known for its Rhône blends, as well as cool-climate varieties like Pinot and Chardonnay. The Central Coast, the largest California AVA, has many different microclimates that lead to a wide range of wines with many sub-AVAs.


text:
If you remove the rubber o ring off the barrel it will cut down the distance considerably.
If you remove the rubber o ring off the barrel it will cut down the distance considerably.
Wayne Nutt
Go Nutts with dog training
HRCH Patton's Parker Co. Shadow "Shadow"
HRCH Clineline Hijacker "Jack"
HRCH Marks a Lot Midnight Hudson, SH "Hudson" RIP (5/26/01-6/18/15)
Castile Creeks Rawhide, MH "Rowdy"
I got streamers from BB years ago, I don't know what kind of streamers you have. Mine are a piece of plastic type fabric white on one side black on the other. It's folded in half with a grommet in the center. I untied the knot at the end of the rope and threaded it through the rope, slid it up against the bumper then tied an overhand knot just above it and it has been there since. Later I added a similar streamer in flourescent orange with a grommet the same way to help me find the unretrieved bumpers in the cover.
The spacer sounds right. I've always used yellow loads and the spacers. It depends how far the mark is but sometimes the light loads can't be heard as well.
I'll add in case you haven't thought of it yet, when you need the bumper to land in a precise spot, just in the cover or short of it or just beyond that bush. Put the BB at the fall and shoot a bumper "backwards to the gun station." Then go set the BB up where the bumper landed. Perfect...
Last edited by John Lash; 05-12-2013 at 09:28 AM.
John Lash
"If you run Field Trials, you learn to swallow your disappointment quickly."
"Field trials are not a game for good dogs. They're for great dogs with great training." E. Graham
After a while the original bb streamers (groumet) broke or wore out. I order replacement streamers from gundogsupply. They are similar in size to the BB ones but are cloth not plastic coated. They come with a groument and a hook (similar to what comes with rabies tags). You can bend it around the rope behind the knot. They are pretty cheap as I recall.
However, we still do not have a description of the dummies you have. Can you post a picture?
Wayne Nutt
Go Nutts with dog training
HRCH Patton's Parker Co. Shadow "Shadow"
HRCH Clineline Hijacker "Jack"
HRCH Marks a Lot Midnight Hudson, SH "Hudson" RIP (5/26/01-6/18/15)
Castile Creeks Rawhide, MH "Rowdy"
Only if you are training in a vacuum where there is NO wind and you have shells that are exactly the same; 2 conditions that NEVER exist in the real world.
On marks where exact placement is critical, no mechanical thrower meets the bill Accurate, experienced live bird boys are essential in those situations
If I get this right, you'll have a picture of the bumpers. The Bumper on the bottom is the "new one" from Bumper Boy. I added the streamers. The one on top is the bumper I got from Gun Dog Supply. They are both about 7" long with identical inserts. The GDS bumper compressed after a few shots (I put the black tape on for visibility). That's why is is a little shorter, now. It's covered with canvas, and I don't expect it to hold up for a very long time. It's already showing some dents and snags from dog teeth. The BB bumper is covered with what appears to be heavy-duty nylon, and is holding up well. I've probably launched these bumpers less than 15 times each, and they are already soiled. I had 4 other bumpers with the streamers attached, and even going down to the lighter green loads, they still blew off at the launcher. These bumpers are just too light. In Kansas, the wind blows, and even with light loads, they carry. I Launched them into the wind with a green load and a 2" spacer, and the bumpers landed 20 yards behind the launcher. I'm still going to order at least one more 4-shooter, but it kind of irritates me that they still show the original bumpers for sale. Seems a bit like false advertising to me. I'm going to keep on experimenting to see if I can come up with something that will work better. I'm thinking about covering one of the bumpers with a thick coat of liquid latex to add some weight. Still don't know about attaching the streamers. I'm thinking that, if I attach them to the front of the bumper instead of the rear, they might stay on. We'll see...
"If the dawg ain't havin' fun, I ain't havin' fun."
Blackkettle's Maximum Overdrive, JH - "O.D." - 1/30/11 -
Blackkettle's Save the Last Dance for Me - "Tango" - 3/10/10
Cottonwood Mallard Hauler, SH - "Moose" - 3/8/99 to 11/20/10
Another problem I see with the new ones is no rope. I sure like being able to tote them all in one hand when I take them out to reload. Trying to carry 8 of those little canvas bumpers isn't going to work without a bucket.
I don't see what the problem was with the old style. I like them and the weight of them. Just now having one that's coming apart and it's only the second one I've had to that going on about 10 years of use. Maybe they'll get wind of the consumers dis-satisfaction and change back.
I bought one of the first BB units, and have upgraded twice. The original bumpers were canvas. I have had several of the foam bumpers blow apart on me...need new ones but doesn't look like there are any available anywhere??
Those are just like the retriev-r-trainer dummies. At least they should make they 10 or 12 inches long so you can see them better and slow the flight down. It is amazing how things with BB have deteriorated so much over the past year. They took a very good, reliable product, with loyal customers and put it all at risk with the Hawx relationship.
John Lash
"If you run Field Trials, you learn to swallow your disappointment quickly."
"Field trials are not a game for good dogs. They're for great dogs with great training." E. Graham