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Drink-drive Wilkins had alcohol in carRead full article on Drink-drive Wilkins had alcohol in car
Former England footballer Ray Wilkins is given a 10-week suspended prison sentence for drink-driving.
Former England footballer Ray Wilkins is given a 10-week suspended prison sentence for drink-driving.
New England manager Sam Allardyce says the job will be the "greatest challenge" of his career.
England's victory in the 1966 World Cup is now a part of the national legend but the tournament had a lasting impact on the country itself.
Sam Allardyce says his man-management attributes will be an important part of his role as England manager.
England captain Wayne Rooney believes Sam Allardyce is a "good appointment" as manager of the national side.
Sam Allardyce is appointed England manager, signing an initial two-year contract after leaving Sunderland.
Southend United boss Phil Brown would be interested in the England assistant manager's job if he was offered the role.
Manchester United manager Jose Mourinho, Harry Redknapp & ex-England boss Sven-Goran Eriksson back Sam Allardyce as England manager.
Sam Allardyce is the new England boss. Here is his life, and career, in pictures.
An Irish priest who handed Sam Allardyce his first job in football management says he "will say Mass" for the "wonderful" new England manager.
Sunderland boss Sam Allardyce will be named as the new England manager, with confirmation expected on Thursday.
The appointment of a new England manager is "getting close", says the Football Association's chief executive Martin Glenn.
Hull manager Steve Bruce is interviewed by the FA about the England manager job, but Sam Allardyce remains favourite for the role.
Steven Gerrard thinks the FA will "get it right" as Sam Allardyce returns to Sunderland and Hull say there has been no Steve Bruce contact.
Wales jump 15 places to 11th in the new Fifa world rankings, as England and Northern Ireland drop following Euro 2016.
Sunderland say they have given the FA permission to speak to Black Cats boss Sam Allardyce about the position of England manager.
Sam Allardyce would be a good choice for England manager, according to his former boss at West Ham, David Gold.
It is not all "doom and gloom" for the future of the England senior team, says under-19s coach Aidy Boothroyd.
The FA wants England's new manager to build a strong team identity rather than provide a short-term fix.
The manager of a primary school football team applies for the vacant England job, saying he's the "perfect candidate" to succeed Roy Hodgson.
Sheep testicles and toilet races form part of the rise of Sam Allardyce - England's new manager.
Set-piece drills, yoga, long passing - what will England look like under new manager Sam Allardyce?
Which of the contenders for the England manager's job would make you happiest...
From Sam Allardyce to Glenn Hoddle and Arsene Wenger to Eddie Howe, we profile six of the men linked with the England job.
After England's embarrassing Euro 2016 exit, Phil McNulty looks at which players will be part of the future - and who is on the way out?
Rarely has the FA had so much to think about on such a short flight home after national sporting humiliation, writes Phil McNulty.
'We will open Iceland up' - BBC pundit Danny Murphy gives his verdict on England's opponents in the last 16 at Euro 2016.
Roy Hodgson has been criticised during Euro 2016 but England have survived - now he must get it right when it counts or face the consequences, says Phil McNulty.
England number one Joe Hart heads into the final Euro 2016 group game against Slovakia under the microscope, but is he undroppable?
Roy Hodgson's England, Russia's sporting future and Uefa's decision-making all face 48 hours of scrutiny, reports Dan Roan.
Seven months of excitement, nerves and tension come to a head on Thursday when England and Wales finally meet at Euro 2016.
As England and Wales prepare to meet at Euro 2016 on Thursday, test your knowledge in BBC Sport's quiz.
The secret stories and key characteristics that transformed a six-year-old stand-in keeper into the most exciting England striker for a decade.
Marcus Rashford is the sort of character who will shine, not shrink in the surroundings of a major international tournament, writes Phil McNulty.
England manager Roy Hodgson insists captain Wayne Rooney will be a key figure at Euro 2016 and should not be written off.
England's performance against Germany was refreshing and encouraging - but must have been difficult viewing for Wayne Rooney.
Fifty years on, the story of how Pickles the dog found the stolen Jules Rimet trophy before the 1966 World Cup.
Four days after the Paris attacks, Phil McNulty reflects on a night of high emotion as England and France pay tribute to the victims at Wembley.
England's predictable defeat in Spain proved once again that they are not in football's elite, says Phil McNulty.
The starting XI, the formation, the squad places - Phil McNulty assesses the big decisions facing England boss Roy Hodgson.
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- Swimmer Ryan Lochte reveals one of his worst dates - US Weekly
- Get the latest on Dr. Dre's arrest - TMZ
- Despite rumors, Eddie Murphy's daughter is not pregnant - Gossip Cop
- The Vampire Diaries star Michael Trevino steps out for a good cause - Just Jared Jr
- Are Blac Chyna and Kylie Jenner on good terms? - Radar
- Blue Ivy is just as stylish as her mom Beyonce! - Lainey Gossip
- This Narcos star confirms he's not coming back for another season of the show - The Hollywood Reporter
22 October 2012 at 3:38 PM
Drew Barrymore & Will Kopelman: Post-Baby Lunch!
About this photo set: Drew Barrymore rocks a heart shirt while grabbing a bite to eat with her husband Will Kopelman at Three Pickles on Thursday (October 18) in Santa Barbara, Calif.… Read More Here
Photo #: 2742415
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An 18-year-old Mequon man was arrested and charged with second degree sexual assualt of a child after he and a 15-year-old girl admitted to having numerous sexual encounters Jan. 5 to 7.
According to the Mequon police report:
The Mequon man was arrested at the Port Zedler Motel, 10036 N. Port Washington Road, on Jan. 7 after he and the girl admitted to having sexual encounters at the motel and one other location. The girl was taken into protective custody.
A 21-year-old Thiensville man, who was at the motel during the sexual encounters, was arrested by the Ozaukee County Sheriff Department on a felony warrant.
More from News and Features
- Pitbull attack in Mequon leaves rare breed dog near death (3)
- Mequon-Thiensville Police Reports, July 21, 2016
- Mequon-Thiensville Police Reports, July 14, 2016
- Mequon girl going to Japan with Trinity Irish Dancers to give free show in earthquake-affected area
- Mequon residents will be Gathering at the Green July 15 and 16
- Cheel-a-bration bringing cheer to Thiensville July 16
- Mequon-Thiensville Police Reports, July 7, 2016
- New Torah scroll welcomed to the Peltz Center for Jewish Life with parade in Mequon
- Video: Peltz Jewish Center parades new Hakhel Torah
- 'Visionary leader' in Mequon receives Prevent Blindness Wisconsin's SEER award


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Pencil It In: Celebs Love This Slim Silhouette
Celebs are sticking to the straight and narrow -- see the latest red carpet hemline report with style tips for wearing midi dresses and other in-between lengths
Louise Roe's kaleidoscope The Nicole Miller midi Louise Roe wore to Audi's 2014 Golden Globes Weekend event isn't for the faint-hearted. With a mix of geometric patterns, bright colors, and bold lines, this dress shows that modest hemlines can be anything but boring.
SEE NEXT PAGE: Miranda Kerr straps in
SEE NEXT PAGE: Miranda Kerr straps in
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Commercial Drain Unblocking
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Chicken Pot Pie Crescent Bake
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Add aromatic flavors to your dinner table with this herbed pot pie that's filled with chicken, veggies and rice and topped with Pillsbury® Crescent Recipe Creations® dough sheet.
All you need to complete this meal is a simple green salad!
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Let's Talk!
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A place to talk about what's on your mind!
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Dealing with a move to a new city
It involves switching jobs, changing child care, and adding more to your juggle. How did you cope?
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Does your family support you?
Discussion about getting support from one's extended family


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News Clamp's xxxHOLiC: Rō Manga to End in February
posted on 2011-01-07 11:06 EST
19th xxxHOLiC volume to ship with original animation DVD in Japan in March
The Japanese publisher Kodansha has confirmed on its website that Clamp will run the final chapter of the xxxHolic: Rō manga in the March issue of Bessatsu Shonen Magazine on February 9. The 19th book volume of xxxHolic will ship with an original animation DVD in Japan in March.
Del Rey published the 16th volume in North America last October. Del Rey Manga Associate Publisher Dallas Middaugh confirmed in December that his company will continue publishing xxxHOLiC for the foreseeable future.
[Via livedoor, Uri-Sure Matome R]
Update: More background information added.
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this article has been modified since it was originally posted; see change history
The Mike Toole Show - Anthology Chronology
With the release of the newest film in the Project Itoh series, Mike takes a look back at the history of other anime anthologies from the Animatrix to Gotham Knight. ― While I sat there trying to digest the second installment of the Project Itoh film series, Studio 4°C's Harmony (note: it didn't sit well), I kept remembering a conversation I'd had with its director, Michael Arias, way back when he wa...
Genshiken: Second Season GN 8
As Genshiken plows further and further into harem romcom territory, Nick Creamer weighs the pros and cons of putting these characters in an increasingly meta situation. ― Genshiken's claim to fame has always been its ability to capture the unvarnished humanity of its otaku heroes. Whether they were heading to a comics festival or arguing about pairings or introducing one of their members to a new hob...
The List - 7 Anime Beach Episodes Worth Watching
As the scorching summer heat reaches its peak outside, you can crank the A/C and enjoy this Classic edition of The List instead, with seven beach episodes that won't leave you feeling high and dry. ― We're reaching the peak of the summer heat, so it's time to keep it cool with a Classic List installment from yester-year. Take it away, past-Lynzee! Beach, pool, and hot springs episodes usually operat...
So Cute It Hurts!! GN 7
So Cute It Hurts is still likely to send you into sugar shock, even though its final pages take things in a disturbing direction. Rebecca Silverman breaks down the twists and turns. ― As far as cross-dressing characters in manga go, Megumu Kobayashi may be one of the least convincing. Go Ikeyamada has a somewhat checkered history with that (one of her previous series, Uwasa no Midori-kun, has an even...
Answerman - Is California The Otaku Promised Land?
Say like you want to move to Japan to wallow around in awesome anime and manga swag all day, but can't afford it - is California the next best thing? ― James asks: I've noticed that the state of California has a lot of Anime Stores and other shops (like Little Tokyo) for Anime. Would California be the ideal place to live in and have access to acquiring physical Anime, Manga, and other related mercha...
Interview: Blue Exorcist Mangaka Kazue Kato
The action blockbuster Blue Exorcist is back with a brand-new anime in 2017 - we sat down with the original creator of the Blue Exorcist manga to explore her incredible success (and dig up details on the new anime!) ― Kazue Kato, the creator of Blue Exorcist made her N. American comic/anime convention debut appearance at Anime Expo 2016 in Los Angeles on July 4th weekend. She came to meet fans and to...
Den-noh Coil Blu-Ray
Despite taking nine years to be released in English, this unique series about how augmented reality tech could change the world is more timely now than ever. Theron Martin dives in. ― Den-noh Coil is an original anime production and the brainchild of Mitsuo Iso, a name that even dedicated anime fans probably aren't familiar with unless they are steeped in knowledge of key animators. (He has done work...
The X Button - First Impressions: I Am Setsuna
This week: Todd looks at Square Enix's throwback RPG! Plus Gravity Rush 2, Nintendo classics, and a major announcement! ― I'll start this column with some very important news: The X Button is retiring. If all goes as planned, the final installment will go up next Thursday, July 28. I must preemptively fend off rumors that the column's being shuttered because of my controversial stand on the Breath o...
Haven't You Heard? I'm Sakamoto GN 4
Sakamoto walks off into the sunset in this final volume of Nami Sato's breakout comedy manga. But do Sakamoto's adventures come to a surprisingly emotional conclusion instead? ― It's hard to believe, but with this volume of Nami Sato's breakout manga, our time with Sakamoto comes to an end. Whether it was planned this way or not, each volume ends up representing one season of Sakamoto's school life, ...


text: Had not been to this place in over 6 years due to my family moving to another state. We were back in town and went for lunch. We were happy to find the place stil has decent service and good food. We got a Burmese Curry, Pad Thai, and Malaysian Beef They were all good. My wife got a Jasmine... More


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Apr 07, 2005 3:56PM PST
So I have been watching the message boards and stuff, and I have noticed that some people who call themselves hardcore gammers are nothing but self glorified fanboys. A hardcore gammer is someone who loves games a lot, actually more than a lot. It is my belief that if someone can't see that each system has its own advantages and disadvantages is no gammer. I personally own PC, Ps2, Xbox, Gamecube, Gameboy Advanced SP, Nintendo DS, Psp, Nintendo 64, SNES, NES, and Genisis. I am a hardcore Gammer
Comments (1)
WhoaPosted: Sep 08, 2005 12:00AM PST by SupaGokuMan
Guess I'm not hardcore, I only have a PS2. But that's bout all I can afford to spend on gaming.


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With Apple, AAPL -0.12459267778416715% Apple Inc. U.S.: Nasdaq USD104.21 -0.13 -0.12459267778416715% /Date(1469826000272-0500)/ Volume (Delayed 15m) : 26765342 AFTER HOURS USD104.25 0.04 0.03838403224258708% Volume (Delayed 15m) : 968346 P/E Ratio 12.174065420560748 Market Cap 561529626663.413 Dividend Yield 2.1878898378274636% Rev. per Employee 1990500 More quote details and news » investors seemingly can't get enough of a good thing. So they rarely complain about many popular exchange-traded funds' heavy emphasis on the stock (ticker: AAPL).
But it's a form of concentration risk, and arguably a more potent one than the Apple profit juggernaut that recently caused UBS analyst Jonathan Golub to build one Standard & Poor's 500 earnings model with the iPad maker and another without it. By Golub's reckoning, 2011 fourth-quarter earnings for the S&P are likely to have risen 6.6%, year-over-year, with Apple and just 2.8% without it, a huge difference.
Investors should at least stop to realize the extent of their wager on Apple (and some other big tech names) in many well-known ETFs. Take the $33 billion PowerShares QQQ QQQ 0.21742911810749696% PowerShares QQQ Trust Series 1 U.S.: Nasdaq 115.23 0.25 0.21742911810749696% /Date(1469826000294-0500)/ Volume (Delayed 15m) : 21309202 AFTER HOURS 115.32 0.09 0.07810466024472794% Volume (Delayed 15m) : 1076118 P/E Ratio N/A Market Cap N/A Dividend Yield 0.9950533715178339% Rev. per Employee N/A More quote details and news » (QQQ) and the $8.9 billion Technology Select Sector SPDR XLK 0.36732929991356955% Technology Select Sector SPDR ETF U.S.: NYSE Arca 46.45 0.17 0.36732929991356955% /Date(1469826000462-0500)/ Volume (Delayed 15m) : 10543797 AFTER HOURS 46.45 % Volume (Delayed 15m) : 45952 P/E Ratio N/A Market Cap N/A Dividend Yield 1.8196684607104414% Rev. per Employee N/A More quote details and news » ETFs (XLK). These are the two most popular tech ETFs, and their Apple weightings roughly match the 16% in the Nasdaq 100.
But the Select Sector SPDR fund's top five holdings–including other household names like International Business Machines IBM -0.4647704034207102% International Business Machines Corp. U.S.: NYSE USD160.62 -0.75 -0.4647704034207102% /Date(1469826243434-0500)/ Volume (Delayed 15m) : 3262068 AFTER HOURS USD160.62 % Volume (Delayed 15m) : 285478 P/E Ratio 12.994094328937788 Market Cap 153527657671.66 Dividend Yield 3.486489851824181% Rev. per Employee 212467 More quote details and news » (IBM) and Google (GOOG) -- make up 45% of its underlying portfolio. The top five stocks in the Nasdaq 100-tracking QQQ account for about 41% of its holdings.
"The fact that Apple is a good chunk of certain technology ETFs isn't a bad thing to us, but obviously you've got a concentration of risk," says Todd Rosenbluth, ETF analyst at S&P Capital IQ. "If one security sees more volatility, whether it's Apple or anything else, then your ETF is going to see more volatility."
There are some surprising Apple gluttons, like the $392 million iShares Morningstar Large Growth Index JKE 0.2115713239482464% iShares Morningstar Large-Cap Growth ETF U.S.: NYSE Arca 123.15 0.26 0.2115713239482464% /Date(1469825969276-0500)/ Volume (Delayed 15m) : 19425 P/E Ratio N/A Market Cap N/A Dividend Yield 0.8569159561510353% Rev. per Employee N/A More quote details and news » Fund (JKE). At 14%, it has the biggest chunk of Apple of any fund that doesn't specialize in technology, according to Ned Davis Research. "Investors may have exposure to Apple they didn't know about," says Neil Leeson, the firm's ETF strategist. In total, there are 19 U.S. exchange-traded funds carrying a weight of 10% or greater in the iPad maker's stock, data from industry researcher XTF.com show.
Fortunately, some tech ETFs avoid a heavy emphasis on any single stock. The downside: Their fees can be higher, and their bid-ask spreads can be wider. Not to mention that investors miss out on the world-beating performance that Apple is currently enjoying.
The $113 million Rydex S&P 500 Equal Weight Technology RYT -0.42603784801347466% Guggenheim S&P 500 Equal Weight Technology ETF U.S.: NYSE Arca 100.5 -0.43 -0.42603784801347466% /Date(1469825930616-0500)/ Volume (Delayed 15m) : 38865 P/E Ratio N/A Market Cap N/A Dividend Yield 0.9996696517412935% Rev. per Employee N/A More quote details and news » ETF (RYT) spreads its exposure pretty evenly among the more than 70 technology stocks, with none grabbing more than a 1.9% weighting as of midweek. But the fund's 0.50% expense ratio is nearly three times what the Select Sector SPDR costs. The $182 million SPDR Morgan Stanley Technology MTK -0.09406215121543743% SPDR Morgan Stanley Technology ETF U.S.: NYSE Arca 56.93 -0.0536 -0.09406215121543743% /Date(1469825384382-0500)/ Volume (Delayed 15m) : 12737 P/E Ratio N/A Market Cap N/A Dividend Yield 1.0070261724925347% Rev. per Employee N/A More quote details and news » ETF (MTK) has no more than 4.3% tied up in any of its 36 holdings. But it, too, has a 0.50% expense ratio.
Meanwhile, there are also pretty substantial differences in the cost of buying and selling shares. The QQQ's bid-ask spread averages are among the tightest in the market whereas the Rydex and Morgan Stanley alternatives are closer to the middle, XTF data show.
Active managers may have had a hard time outperforming the market in 2011 without calling Apple correctly. But they have a choice. Index-tracking ETFs don't have that luxury. They're staking a big chunk of their future on Apple's continued success, with little or no ability to change course.
E-mail: email@example.com


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Grumpier Old Men is a 1995 romantic comedy film, and a sequel to the 1993 film Grumpy Old Men. The film stars Jack Lemmon, Walter Matthau, Ann-Margret, and Sophia Loren, with Burgess Meredith, Daryl Hannah, Kevin Pollak, Katie Sagona, Ann Morgan Guilbert. Grumpier Old Men was directed by Howard Deutch, with the screenplay written by Mark Steven Johnson and the original music score composed by Alan Silvestri. The film was Meredith's final motion picture appearance. He was already suffering from Alzheimer's disease and had to be gently coached through his role in the film. Wikipedia
(after Ariel kicks John out, and Max won't let him spend the night)
John Gustafson: I'm cold.
Max Goldman: (hands him some matches) Here's some matches. Set yourself on fire.
|10 points
|7 points
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Jul 25, 2016 @ 10:38 AM
Viola Davis's Changing Looks
1. 2002
The Tony Award winner made the transition to the big screen with Antwone Fisher and Solaris-and sported a mane of ringlets at the Analyze That premiere.
2. 2004
Long straight locks with plenty of volume for an N.Y.C. event.
3. 2006
Davis rocked a layered cut with subtle highlights at the premiere of The Architect.
4. 2008
She played up her eyes with navy shadow for a screening of Doubt.
5. 2009
The Oscar nominee opted for retro curls at the awards ceremony.
6. 2009
Davis wore her sexy short cut swept over one eye at the premiere of Law Abiding Citizen.
7. 2010
Having scored her second Tony for Fences, Davis framed her face with a chic layered bob.
8. 2011
At the premiere of The Help, Davis paired lush lashes with a dramatic brick lip hue.
9. 2011
Davis debuted fresh blunt fringe at the premiere of Extremely Loud & Incredibly Close.
10. 2012
Davis highlighted her eyes with gilded shadow for the SAG Awards.
11. 2012
"My husband wanted me to take the wig off," Davis told us of her natural look. "He said, 'If you want to wear it for your career, that's fine, but in your life wear your hair. Step into who you are!' It's a powerful statement." For the Oscars, the actress tinted her locks a deep auburn.
12. 2013
For the 2013 Hollywood Film Awards in Beverly Hills, Calif., Davis accented her cropped 'do with a bold lip, a smoky eye, voluminous lashes, and colorful drop earrings.
13. 2014
The actress went for a retro hairdo with glamorous soft waves, accented with a charcoal liner and a nude lip, for the 2014 Academy Awards.
14. 2015
Davis opted for a wavy bob, smoky eye, and a crimson lip at the Disney ABC Television Group's 2015 TCA Summer Press Tour in Beverly Hills, Calif.
1 of 14
The Tony Award winner made the transition to the big screen with Antwone Fisher and Solaris-and sported a mane of ringlets at the Analyze That premiere.
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 10, 1987
|Filing date
|Apr 28, 1983
|Priority date
|Apr 28, 1983
|Fee status
|Publication number
|Anthony J. Sinskey, Graham C. Walker, Kanji Higashio, Eswara A. Rao, William G. Shanabruch, Makoto Yoshihama
|Original Assignee
|Massachusetts Institute Of Technology
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (7), Non-Patent Citations (3), Referenced by (22), Classifications (15), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
______________________________________Enzyme # of Cleavage Sites______________________________________EcoR I 0Hind II 2Bgl II 1Bamh I 1Xba I 0Bcl I 1.______________________________________
______________________________________Enzyme # of Cleavage Sites______________________________________ECoR I 1Hind II 1Bgl II 1Bamh I 1Xba I 1Bcl II 0.______________________________________
______________________________________Enzyme # of Cleavage Sites______________________________________EcoR I 0Hind III 0Bgl II 1Bamh I 1Xba I 1Bcl I 1.______________________________________
______________________________________Enzyme # of Cleavage Sites______________________________________Bcl I 2BamH I 1Bgl II 2.______________________________________
The Government has rights in this invention pursuant to Grant Number NIH-5-T32-CA09258-04 awarded by the U.S. Department of Health and Human Services.
Preparation of protoplasts is well known, especially in gram-positive microorganisms. The technology for protoplast formation in both gram-positive and gram-negative microorganisms is discussed extensively by Hopwood, "Genetic Studies with Bacteria Protoplasts," Ann. Rev. Microbiol. 1981. 35: 237-72. The preponderance of techniques discussed by Hopwood related to protoplast formation in Streptomyces and Bacillus organisms. The transformation of protoplasts is described at pages 263-268. Protoplast fusion techniques are reviewed by Peberdy, Enzyme Microb-Technol., 1980, vol. 2, pages 23-29. Protoplast fusion techniques for obtaining genetic recombination in Brevibacterium flavum are discussed by Kaneko et al. in Agric. Biol. Chem., 43 (5), 1007-1013 (1979). Protoplast fusion in Corynebacterium organisms is described by Katsumata et al. in published Japanese patent application No. 56-109587 (publication date Aug. 31, 1981). The process described by Katsumata et al. involves the use of penicillin and lysozyme. Canadian Pat. No. 1,105,859 also describes protoplast fusion as applied to Streptomyces.
Preparation of "co-integrate" plasmids for transformation of Streptomyces and Escherichia coli is described in U.S. Pat. Nos. 4,273,875 and 4,332,900. EPO application No. 058,889 (Katsumata and Furuya) describes plasmid cloning vectors for Corynebacterium.
The following microorganisms utilized in the invention are available from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A.
A. Corynebacterium glutamicum, NRRL No. B-15340, a species containing the pSR1 plasmid described in Example 1.
B. Bacillus subtilis, NRRL No. B-15305, a species containing the pBD8 plasmid described in Example 2.
C. Bacillus subtilis, NRRL No. B-15306, a species containing the pBD10 plasmid described in Example 2.
D. Bacillus subtilis, NRRL No. B-15341, the species used for transformation in Example 4.
E. Bacillus subtilis, NRRL No. B-15342, a transformed species containing the pHY47 hybrid plasmid described in Example 4.
F. Bacillus subtilis, NRRL No. B-15307, a transformed species containing the pHY416 hybrid plasmid described in Example 4.
G. Corynebacterium glutamicum, NRRL No. B-15302, a transformed species containing the pHY416 plasmid described in Example 6.
H. Corynebacterium glutamicum, NRRL No. B-15303, a transformed species containing the pHY47 plasmid described in Example 6.
I. Corynebacterium glutamicum, NRRL No. B-15304, another transformed (NRRL B-15301) species also containing the pHY416 plasmid described in Example 6.
J. Corynebacterium glutamicum, NRRL No. B-15301, the species used for transformation in Example 7.
Additionally, the invention utilizes the following organisms already deposited with the American Type Culture Collection of Rockville, Md., U.S.A.
A. Corynebacterium glutamicum, ATCC No. 13059, the species used for transformation in Example 5.
FIG. 1 illustrates the scheme for constructing a chimeric plasmid and transforming various organisms.
FIG. 2 is a restriction endonuclease cleavage map for pSR1.
FIG. 3 is the procedure followed to construct the chimeric pSR1/pBD8 plasmid.
FIG. 4 is the procedure followed to construct the chimeric pSR1/pBD10 plasmid.
FIG. 5 is a restriction endonuclease cleavage map for pHY47.
FIG. 6 is a restriction endonuclease cleavage map for pHY416.
By this invention novel plasmids are provided that can be transformed and expressed in at least two different species of bacteria, both of said species being in different genera. The novel chimeric plasmids of the invention can be used to transform Corynebacterium organisms stably and at least one other species from a different genus, i.e., a non Corynebacterium organism. Many plasmids are of narrow host range and can only replicate and express their functions in bacteria of the same genus as the bacterium from which the plasmid was originally isolated. The invention provides chimeric plasmids that can replicate and express their functions in Corynebacterium and at least one organism from a different genus.
The strategy for constructing the chimeric plasmids is first to identify and isolate plasmid DNA from a Corynebacterium organism and then to join that DNA with plasmid DNA isolated from a bacteria of other genera (e.g., Bacillus, Staphylococcus, Streptococcus, Streptomyces, Escherichia, or Pseudomonas). It is most convenient if the plasmids employed in these constructions are relatively small and have molecular weights from about 2.0 to 20.0 Megadaltons ("Md"). Once a plasmid has been identified in a Corynebacterium organism its restriction endonuclease cleavage map is determined by standard techniques. In the early stages of designing such vectors, it is convenient if restriction endonucleases can be identified that cut the Corynebacterium plasmid at single sites so as to minimize the possibilities of deleting or inactivating essential polynucleotide sequences (such as the origin of replication and promoter sequences). If during the construction of chimeric plasmid it turns out that essential Corynebacterium sequences have been inactivated then a different chimeric plasmid can be constructed by utilizing a different restriction site(s) in the Corynebacterium plasmid.
In constructing a chimeric plasmid it is most convenient if the same restriction endonuclease is employed to digest both the Corynebacterium plasmid and the non-Corynebacterium plasmid so that the linearized sequences have cohesive ends. If a different restriction endonucleases are employed to cut the two plasmids then the ends of the plasmid DNA segments may need to be adjusted to permit ligation, e.g., there may be a need to remove unwanted tails, add homologous tails, etc. No matter what technique is used to generate the restriction fragments, the restriction endonuclease sites within the fragments will be preserved so that, after ligation and transformation, the plasmid can be isolated and subjected to restriction analysis as a method for confirming that the desired chimeric plasmid was constructed.
Following digestion, the plasmid restriction fragments from Corynebacterium and the non-Corynebacterium organism are mixed together and permitted to anneal followed by ligation with a suitable ligase, e.g., T4 DNA ligase. The particular ligases employed to ligate specific DNA segments are well known to those skilled in recombinant DNA techniques and include T4 DNA ligase and Escherichia coli DNA ligases. Following construction of the chimeric plasmid, the plasmid can be transformed into Corynebacterium as well as organisms from the genera providing the other restriction fragment(s) incorporated into the chimeric plasmid. Preferably the transformed organisms will be of the same species as the organisms providing the restriction fragment(s) although other species from the same genera as the donating parent species can be employed, e.g., for chimeric plasmids utilizing a restriction residue from C. glutamicum, other Corynebacterium species can be employed as hosts such as C. hydrocarboclastus, C. parvum, and C. michiganense. Bacteria closely related to Corynebacterium such as species of Brevibacterium i.e., Brevibacterium flavum, or B. linens and possibly streptomyces are also candidates. Similarly, if a restriction fragment from Bacillus subtilis is employed, suitable host organisms include Bacillus, Lactobacillus, Staphylococcus, Streptococcus, and Bifidobacterium.
The first step in Corynebacterium transformation is to convert the cells to a form in which exogenous DNA can be taken up by the cells. For example, converting cells to protoplasts could accomplish such a task. In this technique, a part or all of the rigid cell wall of the bacterium is removed so that the exogenous DNA entry can be facilitated with less hindrance. Protoplast formation is accomplished by growing Corynebacterium cells for several generations in the presence of from 0.1% to 2.1% of glycine followed by incubating the cells at from 30�-37� C. for from 0.5 to 4 hours in the presence of lysozyme and (subsequently) treating the cells with a water soluble chelating agent having at least two chelation sites. In addition to lysozyme lipase can also be employed although this is not necessary to prepare protoplasts. Suitable chelating agents include oxalic acid, nitrillotriacetic acid, ethylenediaminetetraacetic acid, and citric acid. The chelating agents are employed in the form of their water soluble alkali metal salts. During protoplast formation the cells are maintained in an osmotically stable (i.e., hypertonic) environment.
Stabilizing buffering agents can be prepared from sugars such as sucrose, ramnose, alcohols such as sorbitol, mannitol and high molecular weight polymers such as PEG and others.
The invention will be further described with reference to a specific embodiment (which is also described in more detail in the examples). With reference to FIG. 1, a procedure for constructing a particular Corynebacterium chimeric plasmid is depicted. Plasmid pBD10 from Bacillus subtilis, NRRL No. B-15306 is obtained. This plasmid (molecular weight about 4.4 Md) has antibiotic markers for resistance to kanamycin ("Km"), chloramphenicol ("Cm") and erythromycin ("Er"). The plasmid is reported to have a single Bcl I site contained within the gene for Em resistance but findings in this invention indicate that is not the case. A second plasmid (designated pSR1-molecular weight about 2.0 Md) was isolated from Corynebacterium glutamicum NRRL No. B-15340. The Corynebacterium plasmid was cryptic, i.e. the plasmid contained no common antibiotic resistance genes but did not contain a single Bcl I site. Following Bcl I digestion, the pBD10 and pSR1 plasmid residues were combined and ligated followed by transformation in to Bacillus subtilis NRRL-B-15341. Twenty-one colonies were obtained that were Kmr, Cmr, and Ems of which 19 exhibited a molecular weight of only 3.2 to 3.4 Md. Two colonies NRRL B-15342, 15307 were obtained showing the same antibiotic pattern but having plasmids with apparent molecular weights closer to the anticipated molecular weight. The plasmid is one of these colonies was designated pHY47 and the plasmid from the other colony was designated pHY416. These plasmids were transformed into protoplasts from C. glutamicum ATCC 13059. The regenerated protoplasts yielded seven colonies from pHY47 that were Kmr and Cms but had a variable molecular weight and were unstable in the absence of Km. Two colonies were isolated from the pHY416 transformation that were Kmr and Cms and had approximately the same molecular weight as pHY416. The plasmid isolated from the pHY416 transformants were used to transform B. subtilis and the resulting transformants were Kmr, Cmr, and Ems. The same plasmid also was used to transform protoplasts from C. glutamicum NRRL No. B-15301 which was resistant to rifampycin ("Rif"). Three colonies were obtained that were all Kmr, Cms, and Rifr NRRL-B-15304.
The chimeric plasmids of the invention represent valuable intermediates for the development of improved host-vector systems. The process for introducing genetic information of interest into plasmids is well known in the art and such processes can be used to insert genes coding for various proteins, e.g., somatostatin, rat proinsulin, interferon, proteases, amino acid biosynthetic enzymes, and antibiotic biosynthetic enzymes. The usefulness of the chimeric plasmids of the invention should be contrasted with procedures for cloning genes into the well characterized Escherichia coli K-12 host-vector systems. The E. coli systems have the disadvantage that genes from some Gram-positive organisms, e.g., Bacillus, do not express well in the Gram-negative E. coli host. Likewise, plasmids from Gram-negative organisms are not easily maintained or are not maintained at all in Gram-positive hosts, and Gram-negative genetic information is either expressed poorly or not at all in Gram-positive hosts. This clearly argues for the advantage of a Gram-positive host-vector system particularly when cloning genes from Gram-positive organisms and suggests the usefulness of plasmids of the invention. Furthermore, since C. glutamicum is not a human pathogen as is E. coli it may prove useful for the cloning of particular genes of medical interest.
The invention can be utilized in several different ways. For example, microorganisms containing the chimeric Corynebacterium plasmids can be obtained from the depositories described above and the plasmids can be isolated from the cells, e.g., the cells can be lysed and the components of the cell wall can be separated from residual cellular components soluble in the lysate. The chimeric plasmid can then be isolated from the lysate. Procedures for accomplishing the above process are well known. For example, the host cells can be lysed by contacting the cells with a suitably hypotonic medium and/or the cells can be grown in the presence of glycine followed by exposing the cells to lysozyme and SDS. Separation of cell wall components can be accomplished simply by centrifugation at from 10,000 to 15,000 rpm, and the plasmid can be isolated from the lysate by cesium chloride/ethidium bromide density gradient centrifugation. The plasmid fraction can be separated from the chromosomal DNA and other components by use of a syringe.
Once isolated the chimeric plasmid can be subjected to restriction mapping followed by digesting with an appropriate set of restriction endonucleases. At this point the genetic information to be cloned can be inserted into the plasmid utilizing well known techniques. For example, the digested plasmid can be treated by well known techniques to make the ends cohesive with those of the DNA fragment containing the gene to be inserted. The plasmid residue is mixed with the DNA fragment carrying the gene of interest, and the plasmid and gene sequences are allowed to anneal, followed by ligation. Subsequently, the plasmid modified by insertion of the new polynucleotide sequence can be used to transform either a Corynebacterium species and/or bacteria from a second genus which contributed a plasmid fragment forming part of the chimeric plasmid. Transformation is accomplished by treating the host cells to render them competent followed by mixing the plasmid with the host cells and incubating the mixture to allow the plasmid to transform the host cells. Where the host cell is a Corynebacterium organism the cell is made competent by forming a protoplast as described above. Protoplasts are then transformed by mixing them with the chimeric plasmid carrying the inserted polynucleotide sequence while maintaining an osmotically stable environment. The mixture is incubated for a sufficient time to allow for transformation of the protoplast, e.g., from 5 to 90 mins at 37� C. The transformed protoplast is then incubated to allow regeneration of the cell wall. Incubation generally requires from 24 to 48 hours at 30� to 37� C. and is carried out in an osmotically stable environment as described above.
The invention can also be utilized by constructing the chimeric plasmid from plasmids obtained from Corynebacterium and from at least one other species of microorganism, e.g., a Bacillus species. The chimeric plasmid is constructed by digesting the Corynebacterium plasmid with a restriction endonuclease; digesting the plasmid obtained from the non-Corynebacterium organism with a restriction endonuclease; if necessary, treating one or both of the plasmid digests to make the ends of the fragments complementary, mixing the restriction endonuclease digestion products of the two plasmids and incubating in the presence of a ligase. The constructed chimeric plasmid can be isolated as described above and further digested with a restriction endonuclease to allow insertion of a foreign polynucleotide sequence coding for a desired protein, e.g., an antibiotic biosynthetic enzyme, amino acid biosynthetic enzyme, somatostatin, etc. The resulting chimeric plasmid carrying the inserted genetic information can then be used to transform a Corynebacterium protoplast or cells of a competent non-Corynebacterium organism from the genera (and preferably species) supplying one of the ligated plasmids.
Suitable Corynebacterium for use in the invention include, but are not limited to, the following species: C. glutamicum, C. fasceans, C. hydrocarboclastus, C. michiganense, C. callunae, C. acetoacidophilum, C. parvum C. pseudotuberculosin, C. granulosum.
Suitable non-Corynebacterium organisms for use in the invention include, but are not limited to, the following: Brevibacterium, Bifidobacterium, Bacillus, Peptostreptococcus, Staphylococcus, Clostridium, Streptococcus, Escherichia, Lactobacillus, Pseudomonas, Streptomyces.
Cell lysis of C. glutamicum NRRL No. B-15340 was performed by a modification of the procedure described by Schiller et al., Antimicrobial Agents and Chemo. 18, 814 (1980) wherein log phase cultures (150-200 Klett units) were treated with 10 ug/ml of penicillin G at 37� C. for 2 hours. The cells were harvested and washed with Tris-HCl buffer (10 mM, pH 8.2). Subsequently, the cells (suspended in 0.3 ml 10 mM Tris-HCl in 0.5M sucrose at pH 8.2) were treated with 5/mg/ml of lysozyme at 37� C. for 1 hour with shaking and, following collection, the cells were treated with an aqueous solution of sodium dodecylsulfate at a final concentration of 1% in 10 mM Tris-HCl, 10 mM EDTA, pH 8.0 (i.e., "SDS") at room temperature for 15 minutes with occasional mixing to lyse the cells. Cellular debris and chromosomal DNA were removed by centrifugation for 15 min at 15,000 rpm and plasmids were then isolated from the cleared cell lysate by CsCl-EtBr density gradient centrifugation as described by Radloff et al., Proc. Natl. Acad. Sci. USA, 57, 1514 (1967). The lower band containing the plasmid DNA was collected by puncturing the centrifuge tube with a syringe needle. EtBr was removed by extraction with isopropanol saturated with 5M NaCl in TRIS-EDTA (10 mM:1 mM) buffer, pH 8.5.
Plasmid DNA was precipitated with 70% ethanol at -20� C. over a period of 2 to 3 days. The precipitate was first washed with 70% EtOH and then with absolute EtOH. The precipitate was suspended in Tris buffer containing 10 ug/ml of RNase (i.e., ribonuclease-A from bovine pancreas). Agarose gel electrophoresis was carried out as described by P. Sharp et al., 1973 at 40 V for 14 hours, and indicated that C. glutamicum NRRL No. B-15340 contained at least one plasmid.
Further purification of plasmid DNA (necessary for restriction mapping) was achieved by subjecting the plasmid DNA to agarose gel electrophoresis through a single slot comb as described by Tabak and Flawell, Nucleic Acid Research, vol. 1 (No. 7, 2321-2332 (1978), i.e., small troughs were cut in the gel first in front of the plasmid band; the troughs were filled with hydroxyapatite suspended in electrophoresis buffer; and electrophoresis was continued for 1-2 hours until the plasmid DNA band had passed into the troughs. The hydroxyapatite-bound DNA was removed from a particular trough, laded on a Sephadex G-50 column and was eluted with 1M potassium phosphate buffer (pH 6.8). The plasmid DNA was precipitated with absolute ethanol at 0� C. Two separate DNA fractions were obtained indicating the presence of two plasmids for ATCC 19223.
The molecular weights of the C. glutamicum plasmids were determined relative to the standard migration patterns of publicly available plasmids, e.g., pBR322 (2.8 Md), pMB9 (3.6 Md), pSC101 (5.8 Md) and pRC1 (8.7 Md) during electrophoresis on an agarose gel. Based on this method the molecular weights of the two plasmids from C. glutamicum NRRL No. B-15340 appeared to be approximately 2.0 Md and 80 Md, respectively. The very large plasmid was not characterized.
Restriction mapping of the 2.0 Md C. glutamicum plasmid was conducted by digestion with restriction endonucleases obtained as commercial preparations from Bethesda Research Laboratories. Enzyme digestions were prepared in accordance with the conditions specified by the suppliers using at least a two-fold excess of endonuclease. The methodology employed was the double restriction endonuclease digestion described by Gryczan et al., J. Bacteriology, 134, 318 (1978), i.e., after incubation with the first restriction endonuclease, the sample was heated at 65� C. for 5 minutes and buffer components were added to bring the composition of the buffer as close as possible to that desired for the second restriction endonuclease. The average molecular weight of the fragments was determined by electrophoretic migration on an agarose gel and comparison with the migration of fragments from Hind III digestion of lambda phage DNA. The sum of the plasmid fragments was approximately 2.0 Md. This data was then used to construct the restriction map of FIG. 2. The sensitivity of the 2.0 Md plasmid to restriction endonucleases is as follows:
______________________________________Plasmid Sensitivities to Restriction Endonucleases# Cleavage Sites # Cleavage SitesEnzyme 2.0 Md Plasmid Enzyme 2.0 Md Plasmid______________________________________Alu I 2 Xho I 0EcoR I 0 Tac I 2BamH I 0 Sst I 2Hae III 2 Sst II 1Hind III 2 Hinc II 3Sal I 0 Bgl II 1Hinf I 3 Bcl I 1Ava I 4 Xba I 0______________________________________
In the remaining examples the 2.0 Md plasmid is generally referred to as "pSR1."
While plasmids can be isolated as described above, a preferable method is the sodium dodecylsulfate/NaCl technique described in Example 2. It should be noted that the 2.0 Md plasmid did not appear to contain genes for resistance to antibiotics, i.e., in this sense the plasmid was cryptic.
B. subtilis was grown in Luria Bertani broth, i.e., ("LB") at 37� C. with shaking. The composition of the LB medium was: Bacto-tryptone 10.0 g; yeast extract 5.0 g; NaCl 10.0 g and 1000 ml of H2 O. Plasmids were isolated by the sodium dodecylsulfate/NaCl method of Guerry et al., "General method for the isolation of plasmid deoxyribonucleic acid," J. Bacteriol., 116, 1064-1066 (1973) followed by dye-buoyant density centrifugation. This technique involves overnight culture in LB broth with Chloramphenicol (Cm), Kanamycin (Km), and Erythromycin (Em). The concentration of all antibiotics was 5 ug/ml. The cells were collected by centrifugation at 6000 rpm for 20 minutes, washed with Tris buffer, sedimented, and suspended in 1/10 volume of a lysing buffer composed of 0.1M NaCl, 0.05M Tris and 25% sucrose, pH 7.5. Lysozyme was added at 0.5 mg/ml followed by incubation at 37� C. for 15 minutes and addition of 4.8 ml/20 ml of 5M NaCl; 1.2 ml/20 ml of 0.5M EDTA; and 26 ml/20 ml of 2% SDS 0.7M NaCl solution. Following gentle inversion the suspension was placed on ice for 18 hours at 4� C. followed by centrifugation at 15,000 rpm for 30 minutes. Cesium chloride-ethidium bromide dye buoyant density gradient centrifugation was employed to isolate the plasmids which were recovered and extracted three times with isopropanol to remove EtBr. The plasmids were precipitated with 70% EtOH; washed once with 90% EtOH; and washed once with absolute EtOH. The plasmids were placed in Tris buffer containing RNase and stored at -20� C.
Two plasmids were identified and given the designations pBD-8 and pBD-10. Restriction analysis and molecular weight determinations for these plasmids were reported by Gryczan and Dubnau, "Construction and properties of chimeric plasmids in B. subtilis," Proc. Nat. Acad. Sci. USA, 75, 1428-1432 (1978) and are set forth in the following table.
__________________________________________________________________________Plasmid Marker M.W. (� 10) EcoR I Hind III Bgl II Bamh I Xba I Bcl I__________________________________________________________________________pSR-1None 2.0 0 2 1 1 0 1pBD-8Km, Cm, Sm 6.0 1 1 1 1 1 0pBD-10Km, Cm, Em 4.4 0 0 1 1 1 1__________________________________________________________________________
Two deposits of the Bacillus subtilis strain have been made in connection with this application. Bacillus subtilis, NRRL No. B-15305, is known to contain the pBD8 plasmid while Bacillus subtilis, NRRL No. B-15306, is known to contain the pBD10 plasmid.
A. A chimeric plasmid was prepared from pSR1 and pBD8. The methods for digestion of plasmids and ligation of digested DNA were those reported by Gryczan et al. in the 1978 reference cited above, i.e., plasmid DNA was suspended in Tris buffer, admixed with Hind III, and incubated at 37� C. for 3 hours. The final concentration of the restriction endonuclease treated plasmid DNA in each reaction mixture was 15 ug/ml. The reaction mixture was heated for 15 minutes at 65� C. to inactivate the enzyme and ligation with T4 DNA ligase was carried out at 10� C. for 20 hours.
B. The pSR1/pBD10 chimeric plasmid was prepared as described by Gryczan et al. by suspending plasmid DNA in Tris buffer followed by digestion with BclI at 50� C. for 3 hours. The reaction mixture was heated at 70� C. for 30 minutes to inactivate the enzyme followed by ligation at 10� C. for 20 hours with T4 DNA ligase.
The pSR1 plasmid has two Hind III sites but does not appear to contain markers for common antibiotics. The pBD8 plasmid contains markers for Kmr, Cmr and Smr. However, the Smr site also contains a single Hind III site and is inactivated during digestion. Transformants containing this chimeric plasmid can be selected for resistance to Km and Cm and sensitivity to Sm although, as discussed below, the Cm gene is apparently not expressed in transformed C. glutamicum hosts.
The pSR1 plasmid has a single Bcl I site and pBD10 has a single Bcl I site. The pBD10 plasmid contains markers for Kmr, CMr and Emr. However, the Emr gene contains the Bcl I site and is inactivated during digestion. Therefore, transformants, in principle, can be selected for resistance to Km and Cm but sensitivity to Em; however, as indicated above, the Cm gene is apparently not expressed in regenerated C. glutamicum protoplasts.
The schemes for construction of the chimeric pSR1/pBD8 and pSR1/pBD10 plasmids are set forth in FIGS. 3 and 4, respectively.
Competent cells of B. subtilis NRRL No. B-15341 were prepared as described by Gryczan et al., "Construction and properties of chimeric plasmids in B. subtilis," Proc. Nat. Acad. Sci., USA 75, 1428-1432 (1978); i.e., after culturing overnight in SPI medium
SPI medium:
0.02% Casamino acids
0.1% Difio Yeast Extract
0.5% Glucose
0.1% Sodium citrate
0.02% Mg2 SO4 7H2 O
at 30� C., the cells were diluted with fresh SpI medium to 10-15 Klett units and incubated at 37� C. until the early stationary phase was obtained. The medium was diluted ten fold into SPII medium (SPI medium plus 5�10-4 M CaCl2 and 2.5�10-3 M MgCl2) followed by incubation at 37� C. for 90 minutes. The cells were collected at 0�-4� C. by centrifugation and resuspended in 0.1 volume of the same supernatant.
The cells were transformed with the chimeric pSR1/pBD8 and pSR/1/pBD10 plasmids prepared as in Example 3. The method employed is described by Gryczan et al. (supra). In the transforming medium the concentration of chimeric plasmid was 15 ug/ml. The transformation medium was incubated at 37� C. for 20 minutes. Transformants were selected using drug plates by the overlay method. As a control, cells were also transformed with just pBD8 or pBD10.
Transformation results are presented in the following table.
______________________________________ Transformants perPlasmid Selected Marker g of Plasmid DNA______________________________________pBD8 Km, Cm, Sm 6.7 � 102pSR1/pBD8 Km, Cm 4.0 � 10.sup.pBD10 Km, Cm, Em 3.7 � 102pSR1/pBD10 Km, Cm 1.3 � 102______________________________________
The transformants were grown for 1 day on LB medium and were screened for drug resistance and the presence of plasmid DNA. Plasmid DNA was isolated by the SDS/NaCl technique (Example 2) followed by purification over agarose gel (Example 1) and restriction mapping.
Analysis of the plasmids indicated that all ten pSR1/pBD8 transformants contained a 5.8 Md plasmid which is considerably smaller than the 7.8-8.0 Md plasmid expected. These clones were all Kmr, Cmr and Smr, perhaps indicating splitting out of the pSR1 fragment. The pSR1/pBD10 transformants yielded two clones containing a plasmid with an apparent molecular weight of 5.2-5.5 Md which approximates the 6.4 Md plasmid expected. Sixteen of the remaining clones contained plasmids ranging from 3.7 to 3.9 Md and appeared to be resistant to Km, Cm and Em, i.e., the improper antibiotic pattern perhaps indicating splitting out of the pSR1 fragment.
The two 5.2-5.5 plasmids from pSR1/pBD10 (designated respectively as pHY47 and pHY416) were subjected to restriction mapping with the following results:
______________________________________Fragment Size Digested (� 10 dal.)Plasmid Bcl I BamH I Bgl II______________________________________pHY47 1.86(2) 6.17(1) 1.86(2) 3.94 3.94pHY416 1.86(2) 6.17(1) 2.02(2) 3.394 3.63pBD10 3.85(2) 4.4(1) 4.52(1)pSR1 1.88(1) -- --______________________________________ () = number of restriction enzyme sites detected.
It is apparent that digestion with Bcl I yielded 2 fragments with molecular weights approximately equal to those of pBD10 and pSR1, indicating that the original ligation points were preserved in the chimeric plasmid. The original pBD10 and pSR1 have unique single sites for Bgl II. Therefore, it was expected that the chimeric plasmid would have two Bgl II sites. Bgl II digestion resulted in 2 fragments as expected but the molecular weights of the resulting fragments from the two plasmis were different indicating perhaps a difference in orientation of the plasmid DNAs during ligation.
Restriction maps for pHY47 and pHY416 are set forth in FIGS. 5 and 6, respectively. The B. subtilis species containing the pHY47 and pHY416 plasmids have been deposited with the Northern Regional Research Laboratory in Peoria, Ill., and bear (respectively) accession numbers NRRL B-15342 and B-15307.
Protoplasts from C. glutamicum ATCC 13059 were prepared by the following method. Cells were cultured overnight in LB broth at concentrations of from 0.7 to 2.1% of glycine. It was found that 2.1% glycine gave the most efficient protoplast formation. The cells were harvested and washed with Tris-HCl buffer (10 mM, pH 8) and suspended in Tris-Sucrose TSSCM buffer (Tris 10 mM, sucrose 0.5M, NaCl 1.0 mM, CaCl2 0.5 mM and MgCl2 25 mM) at pH 8. A 0.5 ml cell suspension was prepared to which 0.5 ml of lysozyme in the above buffer (5 mg/ml) was added followed by mild shaking at 37� C. for 3 hours. EDTA (100 mM) was added followed by gentle shaking at 37� C. for from 10 to 15 minutes.
The extent of protoplasting was followed by plating the cells on LB agar using distilled water as a diluent and comparing the number of colonies formed with those formed when cells were plated on DM3 medium (Chang and Cohen, 1979, Mol. Gen. Genet. 168, 111-115).
DM 3 Regeneration Agar
(Sterile solutions)
4% Agar-200 ml
1M Sodium succinate pH 7.3-500 ml
5% Difco casamino acids-100 ml
10% Difco yeast extract-50 ml
3.5% K2 HPO4 and 1.5% KH2 -PO4 -100 ml
20% glucose-25 ml
1M MgCl2 -20 ml
2% BSA (filter sterilized)-5 ml
Gelatin 5 gm/li
Trace elements:
FeSo4, 7H2 O--5 ug/li
MnSO4, 4H2 O--5 ug/li
ZnSo4, 7H2 O--2 ug/li
using Tris-sucrose buffer (pH 6.5) as diluent. An increase in plating efficiency as a result of exposure of the protoplasts to an osmotically stable environment rather than distilled water was used as an indicator of protoplast formation. The frequency of regeneration of the protoplasts was followed by diluting the protoplasts in distilled water or sucrose buffer (pH 6.5) and plating on LB or DM3 plates, respectively.
The efficiency of regeneration of protoplasts was found to be dependent on the glycine concentration in the original growth medium. At 0.7% glycine concentration, there was over 99% protoplast formation and very high (100%) regeneration of the protoplasts. At higher glycine concentrations, i.e., 2.1%, in the original growth medium the regeneration of protoplasts was only 30-35%. At a glycine concentration of 1.4% in the growth medium, 60% of the protoplasts regenerated.
Electron microscopy indicated that more cell wall was removed during the protoplasting of cells grown in 2.1% as compared to lower concentrations of glycine. A more complete removal of the cell wall is desirable to remove any hindrance to uptake of plasmid DNA by the cell. A concentration of 2.1% glycine in the growth medium was therefore chosen for the preparation of C. glutamicum protoplasts for transformation.
The transformation of procedure employed was similar to a procedure reported for Streptomyces (Bibb et al., Transformation of plasmid DNA into Streptomyces at a high frequency. Nature, 274, 398-400) except polyethylene glycol 8000 (Mol. Wt. 8000, Sigma Chemical Co., St. Louis, MO) was used rather than polyethylene glycol 1000.
Protoplasts (prepared as in Example 5) were transformed with the pHY47 and pHY416 chimeric plasmids isolated as described in Example 4. The transformation procedure was the same in both cases and, as a control, protoplasts were subjected to the same procedure but in the absence of the plasmid DNA.
A 0.6 ml suspension of protoplasts was pelleted in a bench top centrifuge and the supernatant was discarded followed by gentle resuspension of the cells in the remaining supernatant. This suspension was gently mixed with plasmid DNA (10 ug/17 ul buffer) and 0.5 ml of 40% PEG 8000 in water was added and, after about one minute, a further 0.5 ml of 20% PEG 8000 in water was added and mixed for three minutes at room temperature. About 4 ml of SMM buffer (0.5M sucrose, 0.02M maleate, 0.02M Magnesium chloride, pH 6.5) was added with gentle mixing followed by centrifugation for 15 minutes at 1/2 speed in a bench top centrifuge. Enough SMM buffer was added to bring the volume to 200 ul and the pellet was resuspended.
From the final 200 ul suspension, 50 ul was plated directly onto DM3 plates and incubated at 37� C. for 72 hours to allow for regeneration of the protoplasts. Another 50 ul was serially diluted with 5 ml buffer and plated onto DM3 plates followed by incubation at 37� C. for 72 hours. 100 ul of suspension was maintained at room temperature for 60 minutes and was plated directly onto DM3 plates containing 5 ug/ml of either Km, Cm or Km/CM. In the Km/Cm plates 5 ug/ml of both antibiotics were employed. The additional 60 minute period prior to plating onto antibiotic plates has been reported to increase transformation frequency in Bacillus subtilis, i.e., see Chang and Cohen, "High frequency transformation of Bacillus subtilis protoplasts by plasmid DNAs," Mol. Gen. Genet., 168, 111-115 (1979). Transformations of C. glutamicum containing either pHY47 or pHY416 would be expected to be Km and Cm resistant. The protoplasts employed did not contain plasmids prior to transformation and did not exhibit resistance to Km or Cm.
Approximately 60-70% of the original protoplasts were recovered at the end of transformation. No transformants resistant to both Km and Cm were observed on DM3-Km/Cm plates after 72 hours at 37� C. The C. glutamicum protoplasts exposed to plasmid DNA and regenerated for 72 hrs. at 37� C. on DM3 plates containing no Km or Cm were replica plated onto LB plates containing different concentrations of either Km or Cm. The protoplasts transformed with pHY47 produced 7 transformants which were resistant to 10 ug/ml of Km. Protoplasts transformed with pHY416 produced 2 transformants which were resistant to 10 ug/ml Km. None of these C. glutamicum pHY47 or pHY416 transformants were Cm resistant indicating that the Cm resistance gene was either not expressed or deleted upon transformation into C. glutamicum. All 9 C. glutamicum transformants that were Km resistant and Cm sensitive contained plasmids of the same molecular weight (determined as described above) as the respective plasmid used for the original transformation indicating no possible major deletion had occurred upon transformation into C. glutamicum. To determine if the Cm resistance gene was present but not expressed in C. glutamicum, plasmid was isolated from C. glutamicum transformants and used to transform B. subtilis. All B. subtilis cells transformed with pHY416 isolated from C. glutamicum transformants were both Km and Cm resistant proving that the Cm resistance gene was retained but not expressed in C. glutamicum transformed with pHY416.
The transformation frequency for B. subtilis with pHY416 propagated in C. glutamicum was low, e.g., about 0.5 transformants per ug plasmid DNA. In contrast the transformation frequency for B. subtilis with pHY416 propagated in B. subtilis was 1-2�105 per ug of plasmid DNA. The lower transformation frequency with pHY416 isolated from C. glutamicum may be due to modifications of the plasmid in C. glutamicum.
The stability of both pHY416 and pHY47 plasmids in the C. glutamicum protoplasts was investigated by growing the transformants without Km selection pressure. The results indicate that pHY416 was stable and retained in C. glutamicum as compared to pHY47 which was rapidly lost without Km selection.
Sample cultures of the C. glutamicum transformants containing pHY47 and pHY416 have been deposited and bear (NRRL) accession numbers B-15302 (containing plasmid pHY416) and B-15303 (containing plasmid pHY47).
Protoplasts from C. glutamicum were prepared as described above. The particular strain employed was resistant to rifampycin, i.e., Rif� and has the deposit accession number NRRL B-15301. Transformation of C. glutamicum NRRL B-15301 produced three transformants that were Km and Rif resistant although the transformants were again Cm sensitive. This transformant bears the NRRL accession number B-15304.
|Cited Patent
|Filing date
|Publication date
|Aug 21, 1979
|Aug 10, 1982
|The Upjohn Company
|Hybrid plasmid of pBR322 and Streptomyces plasmid and E. coli containing same
|Mar 11, 1983
|Jan 15, 1985
|Cpc International Inc.
|Process for cloning the gene coding for a thermostable alpha-amylase into Escherichia coli and Bacillus subtilis
|Jan 31, 1983
|Apr 2, 1985
|Microlife Technics, Inc.
|Molecular cloning vectors for use in gram-negative bacteria
|Jun 10, 1982
|Apr 30, 1985
|Ajinomoto Co., Inc.
|Composite plasmid
|Feb 11, 1982
|Sep 1, 1982
|Kyowa Hakko Kogyo Co., Ltd.
|Plasmid pCG1
|Apr 27, 1982
|Nov 3, 1982
|Biogen N.V.
|Bacillus cloning vectors, recombinant DNA molecules, bacillus hosts transformed with them and methods for expressing foreign DNA sequences and producing polypeptides coded thereby
|Aug 25, 1982
|Mar 2, 1983
|Kyowa Hakko Kogyo Co., Ltd.
|Plasmid, process for its preparation and its use as vector
|Hopwood, D. A., Ann. Rev. Microbiol. , 1981, vol. 35, pp. 237 272, Genetic Studies with Bacterial Protoplasts .
|Hopwood, D. A., Ann. Rev. Microbiol., 1981, vol. 35, pp. 237-272, "Genetic Studies with Bacterial Protoplasts".
|White et al., Principles of Biochemistry, McGraw Hill NY, 1968, p. 105, 4th Edition.
|Citing Patent
|Filing date
|Publication date
|Feb 1, 1988
|Aug 28, 1990
|University Of Georgia Research Foundation, Inc.
|Cloning systems for Rhodococcus and related bacteria
|Dec 5, 1989
|Oct 23, 1990
|Massachusetts Institute Of Technology
|Coryneform expression and secretion system
|Jun 4, 1990
|Mar 17, 1992
|Autogen Instruments, Inc.
|Nucleic acid separation method
|Feb 1, 1990
|Feb 9, 1993
|Mitsubishi Petrochemical Co., Ltd.
|DNA fragment containing gene which encodes the function of stabilizing plasmid in host microorganism
|Dec 16, 1993
|Jun 20, 1995
|Ajinomoto Co., Inc.
|Plasmid vectors for expression of genes in coryneform bacteria
|Oct 12, 1993
|Nov 4, 1997
|Van Den Bergh Foods Company, Division Of Conopco, Inc.
|Plasmids replicatable in Bacillus subtilis, E. coli and lactic acid streptococcus bacteria
|Jun 30, 2000
|Sep 17, 2002
|Massachusetts Institute Of Technology
|Methods for producing L-isoleucine
|Jun 23, 2000
|Nov 23, 2004
|Basf Aktiengesellschaft
|Corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins
|Aug 20, 2002
|Jan 17, 2006
|Massachusetts Institute Of Technology
|Methods for producing L-isoleucine
|Dec 16, 2004
|Jul 28, 2009
|Paik Kwang Industrial Co., Ltd.
|Gene variants coding for proteins from the metabolic pathway of fine chemicals
|Aug 20, 2002
|Jan 9, 2003
|Massachussetts Institute Of Technology
|Methods for producing L-isoleucine
|Dec 14, 2000
|Aug 21, 2003
|Jens Lerchl
|Moss genes from physcomitrella patens encoding proteins involved in the synthesis of tocopherols and carotenoids
|Nov 7, 2003
|Apr 13, 2006
|Markus Pompejus
|Corynebacterium glutamicum genes encoding stress, resistance and tolerance proteins
|Dec 16, 2004
|May 31, 2007
|Basf Aktiengesellschaft
|Gene variants coding for proteins from the metabolic pathway of fine chemicals
|Oct 31, 2002
|Jun 14, 2006
|Basf Aktiengesellschaft
|Nucleic acid sequence encoding the OPCA gene
|Jun 27, 2000
|Sep 20, 2006
|Basf Aktiengesellschaft
|Corynebacterium glutamicum gene encoding Hpr of phosphoenolpyruvate:sugar phosphotransferase system
|Oct 31, 2002
|Jul 16, 2008
|Basf Se
|Genes coding for proteins of carbon metabolism and energy production
|Jun 27, 2000
|Jan 12, 2011
|Paik Kwang Industrial Co., Ltd.
|Corynebacterium glutamicum gene encoding phosphoenolpyruvate-protein phosphotransferase
|Jun 23, 2000
|Mar 2, 2011
|Paik Kwang Industrial Co., Ltd.
|Corynebacterium glutamicum gene encoding phosphoenolpyruvate carboxykinase
|Jun 23, 2000
|Mar 9, 2011
|Paik Kwang Industrial Co., Ltd.
|Corynebacterium glutamicum genes encoding proteins involved in carbon metabolism and energy production
|Feb 1, 1989
|Aug 10, 1989
|University Of Georgia Research Foundation, Inc.
|Cloning systems for rhodococcus and related bacteria
|Apr 29, 1991
|Dec 12, 1991
|Autogen Instruments, Inc.
|Improved nucleic acid separation method
|U.S. Classification
|435/320.1, 536/23.1, 435/832, 435/69.1, 435/843, 435/839
|International Classification
|Cooperative Classification
|European Classification
|Apr 28, 1983
Owner name: MASSACHUSETTS INSTITUTE OF TECHNOLOGY, CAMBRIDGE,
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|Sep 4, 1990
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I hope everyone is having a wonderful holiday and gearing up for Christmas this Saturday. I'm in Florida enjoying the heat and time with my family. I am popping in today to tell you about a cool affordable online dressmaking apprenticeship that is being launched by a fellow Philadelphia and Drexel University professor. Sally Seligman is the woman behind Dressmaker's Studio, and her team reached out to me last week to get the word out about her Kickstarter campaign, which only has a few weeks left to make it happen!
It is Sally's goal to be the most extensive online dressmaking course, but like any dream, it requires funding. So, while you're wrapping your presents and baking your cookies, take a moment to check out their Kickstarter campaign. If it interests you, or if you want to sign up, show your support so that Sally can bring this one-of-a-kind class to home sewists everywhere.


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Behavioral Ecology and Sociobiology
, Volume 67, Issue 1, pp 43-51
First online:
Retention of acquired predator recognition among shy versus bold juvenile rainbow trout
- G. E. BrownAffiliated withDepartment of Biology, Concordia University Email author
- , M. C. O. FerrariAffiliated withDepartment of Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan
- , P. H. MalkaAffiliated withDepartment of Biology, Concordia University
- , L. FregeauAffiliated withDepartment of Biology, Concordia University
- , L. KayelloAffiliated withDepartment of Biology, Concordia University
- , D. P. ChiversAffiliated withDepartment of Biology, University of Saskatchewan
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The ability to acquire information about predators allows prey to better balance threat-sensitive tradeoffs by responding only to ecologically relevant predation threats. However, predation risk is highly variable through time and responding to predators that no longer represent a threat would likely prove costly to prey. While a wealth of studies have examined the way in which prey learn, little attention has been paid to retention of acquired information. Recent studies suggest that retention is indeed plastic and shaped by a suite of intrinsic factors such as strength of initial conditioning and individual growth rate. Here, we investigated if the duration of retention of acquired information is influenced by individual behavioral tactics (i.e., 'personality'). We recorded latency to escape an opaque acclimation chamber of juvenile rainbow trout (Oncorhynchus mykiss) as a measure of behavioral tactic. We then immediately conditioned individual trout to recognize pumpkinseed (Lepomis gibbosus) and tested for recognition 24 h or 8 days postconditioning. Our results demonstrate that while shy versus bold trout exhibited no difference in the strength of conditioned response to pumpkinseed odor during conditioning trials or when tested for recognition 24 h postconditioning, there was a significant effect of individual behavioral tactic on the retention of learned predator recognition. While shy trout continued to exhibit a learned response to pumpkinseed odor when tested 8 days postconditioning, bold trout were not different from our pseudoconditioned controls. These data suggest that the behavioral tactic employed at the time of conditioning influences the 'memory window' of acquired information.
KeywordsAcquired predator recognition Memory window Threat sensitivity Learning Rainbow trout Oncorhynchus mykiss
Reference tools
- Title
- Retention of acquired predator recognition among shy versus bold juvenile rainbow trout
- Journal
Behavioral Ecology and Sociobiology
Volume 67, Issue 1 , pp 43-51
- Cover Date
- 2013-01
- 10.1007/s00265-012-1422-4
- Print ISSN
- 0340-5443
- Online ISSN
- 1432-0762
- Publisher
- Springer-Verlag
- Additional Links
- Topics
- Keywords
- Acquired predator recognition
- Memory window
- Threat sensitivity
- Learning
- Rainbow trout
- Oncorhynchus mykiss
- Industry Sectors
- Authors
- G. E. Brown (1)
- M. C. O. Ferrari (2)
- P. H. Malka (1)
- L. Fregeau (1)
- L. Kayello (1)
- D. P. Chivers (3)
- Author Affiliations
- 1. Department of Biology, Concordia University, 7141 Sherbrooke Street West, Montreal, QC, Canada, H4B 1R6
- 2. Department of Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, Canada, S7N 5B4
- 3. Department of Biology, University of Saskatchewan, 112 Science Drive, Saskatoon, SK, Canada, S7N 1E2


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September 7, 2013
Knitting, spinning and natural dyeing.
Seven years!
Mushroom dyed baby sweater
I learned to knit, spin and dye with plants at the John C. Campbell Folk School and now I teach textile arts at a nonprofit and am one of the resident artists at the Little Loomhouse.
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Key feature
Women's swimsuit shorts that completely cover the buttocks and top of the thighs. Comfortable elasticated waistband and flattering vertical seams. Ideal for all water-based activities.
User benefits
Shorts that stay very stable when practising aquatic sports
The buttocks, tops of thighs and lower tummy are well covered.
Quick drying
Polymer-based composition, making it quick to dry
Environmental impact
Life cycle comparison: Women's swimsuit bottoms - sustainability.decathlon.com
Swimsuit type
Bikini - Briefs
Main fabric : 80.0% Polyamide (PA), 20.0% Elastane Lining : 100.0% Polyamide (PA)
Test lab terrain
This product has been tested in use in Hendaye and has been subjected to numerous laboratory tests to guarantee the quality of use for which it was designed: tests on the fabric's elasticity and strength, colour-fastness when rubbed, washed, exposed to sea water, chlorinated water and light, and tests on the product's overall performance after repeated washing.
made for
LEISURE WATER SPORTS use
2 years
Care instructions
Wash your swimsuit after purchase. Then rinse in fresh water after each use. If you want to machine wash your swimsuit, wash at 30°C with very little detergent and no fabric conditioner.
Restrictions on use
Not recommended for regular use in chlorinated water.
Body shape
Tribord designed the REVA shorts especially for women who want to completely cover their buttocks, hips and lower tummy, while remaining perfectly comfortable when practising a sport. These microfibre shorts are ideal for all water sports The shape provides good stability. You will never lose them in the waves! You silhouette is visually slimmer thanks to the vertical seams, which also sculpt the buttocks into an attractive curve. Suitable for all body shapes.
Dry sizing and wet sizing
To ensure a perfect fit in the water, it is important to choose the right size while you are dry because once they are in the water, swimsuits become looser. We therefore recommend you buy the correct size. The swimsuit needs to fit snugly against your body in the fitting room: you should feel supported. The elastane contained in certain components used in the design of our swimsuits softens and relaxes in the water.
Score to compare the environmental impact of products
The environmental impact of the product is calculated over its entire life cycle and with different indicators. An overall score (ABCDE) is attributed to help you easily identify the products with the best environmental performance by comparing products of the same type with one another (T-shirts, trousers, backpacks, etc.). Decathlon chose to apply this voluntary environmental labelling scheme. For more information, visit: http://sustainability.decathlon.com/
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Great fit -
I already had a tankini top but wanted swim shorts to go with it, these were just what I wanted and didn't disappoint when they arrived. Definitely recommend.
Very comfortable, good length Nothing
They are true to size ans comfortable, nice leg coverage to.
comfortable; good fit; good coverage none
I wore these swim shorts over my swimsuit for snorkelling and paddle boarding; the gave good coverage, were a good fit and were very comfortable
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I need more financial aid.
Health & Safety:
I never felt unsafe in Santa Barbara especially on campus.
Overall Experience:
I loved my 1st year residence hall experience. This school offers a bit of everything, social, academics and personal freedom right by the beach.
Although it is not disciplined, it offers me different ways to explore everything I am learning. For example, I can travel and see hand in hand what I am learning about in a book.
They offer a variety of different classes that always challenge you.
Campus Food:
The dinning commons are great. They all have their own style and every dinning common is known for what food entree they are best at.
I can't give a valuable opinion on this.
I love it! It offers a variety of things you can learn and it is not so disciplined.
Local Area:
It's a great college atmosphere with parties and fun, but families also live there which we should all respect.
The bus is a great help to all of UCSB'S students, but it gets paid from our tuition.
Campus Housing:
The cost is very expensive, but it's a great experience.
Off-Campus Housing:
Isla Vista is a fun place to live, but it is expensive since it is beach side
My financial aid experience worked out in the end, but it was a hassle and nerve-racking, but isn't everybody's experience like that?
Greek Life:
Our Greek Life is pretty big, but it doesn't put a wedge between Greeks and Non-Greeks.
Campus Quality:
Many different options, recreation center, the library, UCen, etc.
Campus Quality:
Many of the facilities are beautiful and have easy access, some need to be remodeled.
I am able to manage my major, be in greek life, and have a job.
Engineering department is great for me. Mathematics department could use some work.
Overall Experience:
I absolutely love my school and the people I have met here
Drug Safety:
I don't drink or do drugs, and I have a perfectly normal and fun social and party life. People are very accepting to the option of doing or not doing drugs/ drinking alcohol
free bus rides and they go many places
Guys & Girls:
Many interesting and kind people to get to know
Many different clubs and organizations for people of different sexuality, ethnicity, religious views, etc.
Off-Campus Dining:
Many places to eat in Isla Vista
Have a great program for my major
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Ask practically any UCSB student, and he or she can give you a long list of things they love about the University, along with a few things they wish they could change. For the most part, UCSB is a college student's haven with great academics, world-renowned professors, not to mention the great weather and laid-back atmosphere, and lastly who can forget the social scene? Some students describe Santa Barbara as paradise, but for those who quickly get over it and need a getaway, LA is a short one-hour drive south, while San Francisco is only four hours north. There are hidden treasures in Santa Barbara that you can explore and take advantage of while living there.
Do not let the weather and social scene cloud your judgment and decisions—take a deeper look at the school and ask the students. Chances are, the parties are not at the top of their list. Most students fall in love with their internships, clubs and organizations, and research work that prepare them for the real world. The most successful students are those who find a balance between academics and social life. The temptation to blow off school is present wherever you go, so the students pay extra attention to the work they produce. Overall, UCSB is the type of school that produces people that employers are seeking. You will find your fellow peers to be sociable and well-rounded in and out of the workplace. So although you may not get to experience game days like you would at a football school, there is nothing better than being able to catch a quick "surf sesh" before and after your class.
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Bio: Alias for Now is a Rock n Roll band hailing from Greenville, SC. Putting the "Power" in power trio, Alias for Now always delivers the rawest, loudest, most in your face show anywhere. Taking Sex, Drugs, and Rock n Roll literally, AFN will have you on the edge of your seat screaming for more. Comb...See Full Bio
"Equal parts MC5, and Black Sabbath, this band takes a familiar sound, and makes it their own. There's plenty of guitar-shredding goodness to be had, but not at the expense of the song itself. "Don't Say Anything" has an almost poppy bounce to it before exploding into a thick, heavy groove halfway through the song, complete with Thin Lizzy-style guitar harmonies. This is early heavy metal with a fantastic pop sensibility, a true anomaly in the current musical climate, and a very welcome one. It's no wonder this band has shared the stage with such a wide variety of rock bands, they have a little something for virtually every taste. Not to mention they put on a damn good show."


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Medivet Borehamwood144 Shenley Road
Our branches provide a dedicated local service to clients, offering a comprehensive, quality service at affordable prices. Our branches can also provide access to 24 hour services via our linked Hendon 24hr Continuous Care and Emergency Centre.
As a part of the Medivet family, your pet will have the benefit of the skills and experience of over 200 vets throughout the company. Our staff aim to provide a welcoming and professional environment for both pet and client.
We offer both diagnostic and surgical care as well as a wealth of additional benefits to keep your pet happy and healthy.
Clinic reviews
5 Stars - 08-07-2016 - Kim and Terry
Stuart has been our vet for so many years now. We value and trust him and his team to look after our cat. It's such an excellent practice that we travel 20 miles to get there. Sarah Hall vet nurse has also been a constant and is always very knowledgeable and helpful.
5 Stars - 13-11-2015 - Cheri
We called Medivet in a panic back in July when our kitten came home limping one afternoon, it turned out our kitty had to have extensive surgery on his leg which still needs treatment. Stuart and his team have been amazing, they have not only looked after our pets but us too. Unfortunately we needed to rush our dog in to the surgery recently and I can only say the service received is first rate. Everyone in the surgery is caring and professional. THANK YOU MEDIVET BOREHAMWOOD from Hugo, Ruby and their humans
5 Stars - 14-06-2014 - Angela HS
I have been attending Medivet in Borehamwood with my dogs practically since the day it opened. I once adopted two elderly Bichons who had digestive problems and with Medivet's excellent advice and help we managed to sort them out so that they had a good quality of life. I have always managed to get an appointment, on the same day if necessary, have been treated with courtesy and friendliness, have been fully involved in discussions about any treatment needed and have been consulted regarding any medications prescribed. I trust them completely with the care of my dogs.
5 Stars - 29-05-2014 - Lee Cousins
We cannot praise this service enough for all the consideration and caring we have experienced in the 12 years we have used Dr. Stuart as our vet. He and his staff, especially Sarah, have places in our hearts as very special people. They are blessings. Stuart's genuine compassion, love and respect for our beloved Maine Coon cat when he came to the end of his life, were deeply appreciated, and now he has the care of two more who have joined our family. We'd never trust them with anyone else.
5 Stars - 21-05-2014 - BC
The Vet and Staff are genuinely caring and gentle, and treat my animals as though they were one of their own. The team is professional and helpful and always willing to find solutions.


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Do you remember????
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17,561 - 17,580 of 17,990 Comments Last updated Yesterday
#18486 Oct 26, 2013
HELLO ALL,yes FR i do remember going to the lowell lawrence game.first in the stands,then on the field.i see that there is no more field day.PAUL,VITO and I all marched as boy officers,i'm sure they remember that.i don't think there are no more boy and girl officers.it would be nice to get back to our memories of growing up in lowell.weather up here is getting colder.highs in the forties and lows in the low thirties.we've had this cold weather before halloween.i've got the two new pairs of long johns,and i had the snow blower tuned and we're ready for the snow!how's the weather in lowell?hope to hear from you guys and gals.FR,are you and the missus back down there in florida?SLA,hope you and the family are in good health.all for now.cougar.
#18487 Oct 27, 2013
They did away with the Boy and Girl Officers after 70 something years, I also marched as an officer. We are back in the Sunshine State, don't miss that cold weather at all. I sold my snow blower and lawn tractor now there is no snow here and I watch the maint. crew do the lawn. Lot of shootings in Lowell lately, not like the old days at all. I do miss the people back in Lowell and will visit with them when I return for the Holidays. Take care WSC.
#18488 Oct 28, 2013
FR,back in the day,it was quite an honor being a boy/girl officer.although i became a boy officer more for my ma,then me.i can remember how excited everyone was the week before field day.i was a captain,what rank did you have?i must admit being a boy officer helped me in the navy,as i was an e-3 straight out of boot camp.at the time i was in,most everyone was an e-1 out of boot camp.nice to hear from you,and i too hope we can get everyone back on here without any BS.cougar.
#18489 Oct 28, 2013
Hi,FR,how r u doing?Good,I hope.Can u even believe what has has happened to our cuntry in the past year.?I am absolutely mortified by what this PREZ????? has done to us.You N.E.'ers have to stand up and say ''THIS IS WRONG''No more eating away our childrens legacy.This man??? has got to go.!!!!I'll donate for Michelle or Billary before I send him another nickel.!!!Think about the LIES he's given us in past year. It's absolutely FREAKING amazing,and he is still getting away with it.!!!Yes,I'm from the Lowell area,but I live in Florida now,and relief hounds are not welcome here.!!!If U don't work U don't eat,PERIOD.!!!!!
#18490 Oct 28, 2013
Yes,I do remember Lowell,and if you did'nt work or scam,you did'nt eat or drink.That's how it should be.You folks up there,as much as I love you have gotten too soft for your britches.!!!You might call me a redneck,a hillbilly,a bumpkin,a hayseed,but that's what I am now.And I'm awful glad I did.I've had a wonderful life here,and as much as I miss New England I woundn't trade a second of it.Any of you that don't leave that misery they call winter to come South are crazier than I am.ENJOY.LOL
#18491 Oct 28, 2013
Well,I guess I've gotten political enuff for a site that doesn't get political.I'm sorry,AGAIN,for that.I just don't like the way this country is going.Peeple have to take responsibility for themselves.Don't expect the government to help you in time of need.U must cheat,steal,lie,whatever the F- you gotta do to survive.Dear Mr. O is not gonna help you,he wants to own you.Unnerstand.?Enuff said.? I definitely think so.My poor neck.I think I stretched it to the breaking point.LMAO Luv u guys & gurls.!!!
#18492 Oct 28, 2013
OK,guys,one more more time.We need a good laff in the midst of this Halloween Horror.Watch Toby Keith on U-Tube,with him laying bricks,''It's a little too late.''Good Golly,it says it all.!I hope U all have a Happy,Horrible Halloween.I hate like hell to admit it,but I luv this website.
#18493 Oct 29, 2013
Hey WSC.
How are you.? My has gotten worst is now on Palliative care, Which is followed by Ospis care.
Glad to here from you. How is Sheri?& Princes
Yes it would be nice to carry on this site.
Hope to see you and Janet and Fr back on here.
Along with some others.
#18494 Oct 29, 2013
Florida State Panther.
A/k/a Tommy Boy. How you doing.
How is your Mom doing ?
Hope everything is ok.
Lets get this going again.
And Ignore any Idiots.
#18495 Oct 29, 2013
How are you doing,buddy?I'm sorry to hear about your girl.And who are you calling an idiot? Not me,I hope.And if A/k/a means ''Another Known A-hole,you got it on the button.LOL
#18496 Oct 29, 2013
I'm doing ok. Thanks.
Cold here about 44 and windy. How is your Mom ?
Hope this site keeps going.
We just don't answer idiots.
Frustrate them. Take Care.
#18497 Oct 29, 2013
Well hello there.I was just up in Billerica Mass last weekend for a bridel shower and to see my son and the weather was beautiful,the tree's are so pretty with all thier color.Went to Brown's at the beach the food was great.Now I will wait till you get a lot of snow then visit for a week.I do miss the four season's.Take care.
#18498 Oct 29, 2013
Hi Tom Palm coast is on the East coast right??
#18499 Oct 30, 2013
Hi,Janet.All is well with you,I hope.You know I try to have the utmost respct for girls on this site,but what a dumb question.!Yes,Palm Coast is on the East coast.Don't you own a map.I'll send you one.I have hundreds.We're a big city now,over 50,000 people.Even I know where Teenie Weenie Wachee is,and I don't mean in my pants,and I don't usually need a map to find it.HaHaHa I'm sorry,but I need some lunch,and a nap.Goodnite.
#18500 Oct 30, 2013
TOMMY,what a reply to janet's question.you turned your reply into some kind of sexual reply.i see you did a LOL however,this is the kind of stuff that will back you know who.how's your mom doing these days?good to have you back on the topix.the cold weather should bring back all our friends TOM.it's ten in the morning here,and the temp is twenty-one degrees.we had a trace of snow on monday.when winter comes to the pacific northwest,i really ENVY all you folks who live/winter in florida.anyone who wants a shot of cold weather,let me know i'll send some,ASAP.cougar.
#18501 Oct 30, 2013
Come on. Why you being so rude to Janet.
I see WSC told you the same thing.
Lets get along. There was no need for what you said to her.
I think you should apologies' to Janet.
Its cold enough here I don't need your cold. lol.
Take Care.
#18502 Oct 30, 2013
SLA,your girl is in "good hands"with hospice care.i've been a volunteer with hospice,for many years.you don't need to worry about anything with hospice.if you want hospice will cook meals for you folks,they'll make two weeks supper and freeze them.when you want a meal,just pop one out and microwave your meal.the volunteer's will clean house for you,and if you need some time off,the volunteer's will sit with your girl.great organization,great people.all is well with the family,thanks for asking.all for know.cougar.
#18503 Oct 30, 2013
OK,guys,I get it.I guess nobody in Lowell has a sense of humor anymore.Janet,I'm very sorry,I didn't mean to be one bit disrespectful.I always thought the name of your town was kinda funny.Life gets pretty ugly without a little joke getting thrown in once in a while.I'm not like this in person,but keep the keyboard away from me.I'm very sorry again,Janet.It won't happen again.And I think the pissy,whiny crybabies need a nap,too.!!!!
#18504 Oct 30, 2013
WSC, I liked your note because I became a BO because my Mother wanted me to be one, Capt. then onto Major and our Company came in second place during Field exercises.
This site was going good till Tommy boy got on and started with his foolish typing's. He then writes that he was joking and us NE people cannot take a joke, now who could possibly believe that??
#18505 Oct 30, 2013
Well,I should have expected to hear the 'Third Stooge' put in his two cents sooner or later.Like Gomer said ''Surpise,Surprise,Surprise.! The only woman that didn't complain was Janet.I'm very sorry again,Janet,It was really just another one of my morning rants.I just hope you four girls keep it rolling.I'm unplugging my keyboard when I visit this site,so no worries.OK.? I'm done defending myself.HAGreatDay
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