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VIEWS: 4 PAGES: 1 POSTED ON: 2/18/2011 Public Domain
Fuerteventura is one of the Canary Islands, in Atlantic Ocean. Its believed that, the first native of Fuerteventura have arrived from North Africa. They lived in caves and dwell. By the start of 11th century, the Phoenician settlers arrived in Fuerteventura. In 1405 French conqueror Jean de Be'thencourt conquered the island. The island began to develop since 1859, when 151 year military rule was dissolved and is announced as an entirely new capital. The major breakthrough in the history of the island happened in the year 1912, when it secured the right to self-govern. By 1940s Fuerteventura Island got it own airport. Tourism began to flourish in Fuerteventura by the building of Fuerteventura airport. It's situated in EL Mattoral, 5 km south west of the capital. As the island is close to Africa, many illegal immigrants take the sea way to enter European Union. Fuerteventura Island offers pleasant climate throughout the year. The sea protects the Fuerteventura Island from the hot Sahara winds. A sand storm known a Calima can cause the temperature in the Island to increase up to 10° Celsius. The temperature rise is temporary. The island is also home to two endangered species of Canarian Egyptian Vulture. The first tourist hotel was built in 1965. Fuerteventura is sitting comfortably in the world's stage as a major European holiday destination. It is also known as the surfers paradise, as the summer trade wind and the winter swells of Atlantic make year round surfing possible. Scuba diving big fish lovers are all drawn to Fuerteventura, as it has got it all. Fuerteventura is also packed with long beaches and remote bays. In the north there is Correlejo and El Jable which are made up of fine sand dunes. Most of the surfing takes place in the south side, as there are rarely any beaches in the north. No wonder, the economy of Fuerteventura completely depends on tourism. There are a large number of boats to travel from One Island to another. There are fast ferries, which links almost all the island. Both type of transportation is capable of transporting large number of people and cargos. Fast ferries are normally built of aluminum and they are powered with most modern Diesel engines. There are an 8 airport in total, in the Canary Island group. Fast ferries travel faster than the conventional ferries. The fast ferries can cruise up to 30 knots per hour, whereas the conventionally ferries struggle to even touch the 20knot per hour mark. Most of the people opt for the fast ferries. Road transport constitutes of public transportation system and the hire a car service. As Fuerteventura is a tourist spot, the mast widely used transportation system is hiring a car. Dedicated offices of cheap car hire works throughout the Fuerteventura Island. Car hire services are reliable and comfortable than other means of transportation. Facilities such as prior booking are made available to the tourists. Car Hire Fuerteventura Airport offers the best service for the customers. Car Hire Spain can be used to hire a car anywhere in Spain. Visit Car Hire through which you can choose the car you like for your transportation in Fuerteventura.
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Grow virtual flowers and arrange them in gardens. Pick your favorites and display them on your phone's home screen.
Even better: share pictures of your best flowers with other Daisy Gardeners for them to scan and plant in their own gardens.
DARTSLIVE's official app is now available!
This gives an easy access to your darts performance data and the tools to communicate with your darts pals.
What's DARTSLIVE?
It is a collective term for a network-connected dart machine "DARTSLIVE" and its associated services provided with the use of PC, Smartphone and dedicated IC Card "DARTSLIVE CARD".
This application enables you to view your play data and use the following functions.
Your darts performance data is available for viewing anytime, anywhere.
People with whom you play darts become your "Friends"! Play darts and make more friends.
Send short messages and stickers to your "Friends" for easy and quick communication.
Get the latest DARTSLIVE information!
Dartslive Rating Check
The DARTSLIVE card of the paid member is required.
Can browse in one screen with current Rating and stats, award by today's play.
rating of the next day is presumed and displayed from the play of the day.
* However, it is not an exact value.
Please wait for an exact value till the next day, or confirm it by the TouchLive.
The saved play data can be displayed in a graph.
Theme changes are published separately can done with the "Dartslive Theme Changer".
Can be made to start if "change theme" is selected.
The item which can be checked
Current Rating
Rating of the next day (estimate with error)
Stats / difference on the previous day between 01 and Cricket
Bull rate / D Bull rate
Triple rate
Today's Award number
Today's stats highest, the minimum, and an average
History data of rating stats count-up (graph, list)
01 DART OUT and best score of practice
( A display is changed by horizontally scrolling in the column of Stats.)
Dartslive Theme Changer
All the possessed themes are displayed in a list.
Moreover, the list display can perform a filter with a theme name or theme type etc,
and sorting by a date of acquisition, a theme name.
The change of the setting theme from the app.
Two or more cards are registered and the display to change is possible.
There is an APP original favorite function.
Do not link the thing of the official site, but there is not the setting upper limit.
the favorite setting
It is possible from the menu which is displayed because long tap a theme.
based on the number of the possessed themes,
it takes time till reading completion of a theme at the time of first time registration of a card.
More from developer
Let friends and family easily find out where you are by sharing your PIN number with them: They send an SMS, Pintail replies with your location.
(Fixed for compatibility with the cupcake release.)
Meadow Live Wallpaper
Up to 2,000 individual blades of grass come together under rolling clouds to form this relaxing live wallpaper.
Metaglow allows you to discover, keep and share computer-generated background designs. It uses a genetic mutation to create designs that are styled by the choices you make.
Everyone's favorite designs are automatically shared, allowing you to find designs you like among those that others have discovered.
You can create an account within the application using your Google, Twitter or Facebook accounts. Registration is optional, but recommended to ensure that you can reclaim your favorite designs if you uninstall the application or replace your device.
See the in-app help which is accessible via the menu.
Downloading applications with Betan requires a password from the publisher.
Any developer can use Betan to publish their applications privately. Check out the website for more information.
Daisy Live Wallpaper Lite
It features a beautiful cascade of delicate flowers against a slowly undulating background.
There's a different design for each page of your homescreen.
Touch sensitive, tap the flowers to pop their petals.
Full version has more flowers and lets you pick more from Daisy Garden.

subset: commoncrawl


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id Software's RAGE to use Steamworks and Steam Cloud
View Single Post
Registered User
Join Date: Apr 2007
Location: UK
Posts: 2,523
Re: id Software's RAGE to use Steamworks and Steam Cloud
I really dont see the problem with Origin. Apart from a few people suggesting that they like to have all DD in one place i feel as there is no valid debate here.
I have a GFWL account, Steam account, WoW account and an Origin account with games on them all...when i browse to my 'games' folder each and every game from each distributor is there...all in one place. I dont care for the silly things such as in game chat interface and screenshot taker and crappy fps readouts that are usually inaccurate nor am i interested in 'chatting' on these interfaces.
Usually the prices of games on these DD acounts are usually higher than retail and so if i have an option i will always mail order my games and usually save myself 10-15 quid in doing so. THIS is my only gripe with DD services.
I play a game on Steam...I play a game on Origin... Is there a difference to me when im playing these games on what DD account i have used to acquire the game? No there isnt. Also Steam Cloud can be a huge fail at times as i have personally witnessed lost saves and game resets because of this, personally i like all my data being stored locally.
EA have stood up and said "hold on, why are steam making soo much money off our games? Maybe we should distribute them ourselves with an improved EA downloader". Good on them i say, Steam is becoming overpriced and greedy for AAA titles and at the end of the day you are downloading data...No production cost here for DVD's, manuals and boxes yet they still charge extortionate prices.
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By MICHAEL CAINES
The novelist Isabel Colegate is best known for The Shooting Party, published in 1980, in part because of the film adaptation that quickly followed. Her first novel, The Blackmailer, has a more interesting claim to fame, a story from the small world of mid-century publishing that I believe she's written about before, but which I only discovered from the reissue published last year with a new foreword. . . .


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Federer sails into last eight at HalleRoger Federer kept his chances of an eighth title at Halle on track when he overpowered Latvian Ernests Gulbis 6-3, 7-5 on Wednesday to breeze into Asian Age, 2 weeks ago
Federer eases into quarters - Bangladesh Daily Star, 2 weeks ago
Federer cruises into Halle quarters - Tennis.co.uk, 2 weeks ago
125 images for ernests gulbis
Federer enters Halle Open quarterfinals
Berlin: World number two Roger Federer comfortably beat Latvian Ernests Gulbis 6-3 7-5 on Wednesday to move into the last eight of the Halle Open where he will meet Germany's Florian Mayer. The 17-time grand slam winner, preparing for Wimbledon's ...Manorama Online, 2 weeks ago Federer on course for another Halle ti... IOL, 2 weeks ago Federer through to Halle third round BBC, 2 weeks ago Tennis-Federer cruises past Gulbis into last eight at Halle Yahoo! UK and Ireland Eurosport, 2 weeks ago
More from: ENCA, ENCA...and 25 other sources
Roger Federer advances to quarters in quest for 8th Gerry Weber title
Top-seeded Roger Federer moved closer to a record eighth title at the Gerry Weber Open by beating Ernests Gulbis 6-3, 7-5 on Wednesday to reach the quarterfinals. After fending off the only two breaks points he faced in the third game, one break for ...Austin American Statesman, 2 weeks ago Federer Advances to Quarterfinals of Gerry Weber Open ABC News, 2 weeks ago Nishikori, Monfils advance at Gerry Weber Open MySanAntonio, 2 weeks ago Nishikori beats Brown, advances to Weber Open quarterfinals Japan Times, 2 weeks ago
More from: Miami Herald, MSN UK...and 5 other sources
French Open: Eugenie Bouchard, Ernests Gulbis struggle with comebacks after a year full of surprises
Bouchard, No 6 in the world and last year's Wimbledon finalist, has not seemed the same since she split in the off-season with her coach Nick Saviano. By Christopher Clarey Watching Eugenie Bouchard suffer at the French Open on Tuesday and watching Simona ...Economic Times, 1 month ago On Tennis: Former Breakthrough Players Endure Breakdowns in Paris NewsOnFeeds.com, 1 month ago
Slumping Gulbis a stranger to stress amid disaster season
Laid-back Latvian Ernests Gulbis refused to bow to stress on Monday as the 13th seed exited timidly from the Monte Carlo Masters, a 6-1, 6-0 first-round victim of Austria`s Andreas Haider-Maurer. The admittedly slumping world number 17 suffered his eighth ...Zee News, 2 months ago Ernests Gulbis loses in 1st round at Monte Carlo Masters IBN Live, 2 months ago
Isner into semis; Gulbis falls in Nice
Nice, France (SportsNetwork.com) - Second seed John Isner secured a berth in the semifinals, while defending champion Ernests Gulbis was sent packing Thursday at the Nice Open, a final clay-court French Open tune-up. The 6-foot-10 American Isner ...FOXNews.com, 1 month ago
Sonstiges: Federer im Viertelfinale von Halle gegen Mayer
ATP-Turnier Halle: Federer zieht problemlos in die Viertelfinals ein Roger Federer besiegt Ernests Gulbis 6:3, 7:5 und steht im Rasenturnier in Halle in den Viertelfinals. Dort wartet der Deutsche Florian Mayer.... 20 Minuten | Front - Heute Neu ...Naija Lately, 2 weeks ago ATP-Turnier Halle: Federer und Gulbis im zweiten Satz gleichauf Naija Lately, 2 weeks ago ATP-Turnier Halle: Federer zieht problemlos in die Viertelfinals ein Naija Lately, 2 weeks ago ATP-Turnier Halle: Federer serviert zum Matchgewinn Naija Lately, 2 weeks ago
Tennis: ATP Halle Open results - co …
Results from the third day of the ATP Halle Open on Wednesday (x denotes seeding): 2nd rd Roger Federer (SUI x1) bt Ernests Gulbis (LAT) 6-3, 7-5 Florian Mayer (GER) bt Steve Johnson (USA) 6-3, 7-6 (7/4) Tomas Berdych (CZE x3) bt Borna Coric ...Yahoo! India, 2 weeks ago Tennis: ATP Nice Open results -- 2nd … Yahoo! India, 1 month ago
Isner Reaches Open De Nice Semifinals; Gulbis Goes Out
Second-seeded John Isner of the United States beat Serbia's Dusan Lajovic 7-6 (3), 6-4 to reach the Open de Nice semifinals on Thursday, while defending champion Ernests Gulbis lost 6-2, 6-4 against Austria's Dominic Thiem. Isner, who had eight ...ABC News, 1 month ago Isner moves into Nice semis News24, 1 month ago Isner advances to Open de Nice semifinals ESPN.com, 1 month ago
More from: , Times of India...and 1 other sources
Simon pulls out; Gulbis reaches Nice QFs
Nice, France (SportsNetwork.com) - Top-seeded Frenchman Gilles Simon pulled out of the draw, while third-seeded defending champion Ernests Gulbis was a hard-fought second-round winner Wednesday at the Nice Open, a final clay-court ...Sports Network, 1 month ago
More from: , FOXNews.com...and 1 other sources
Realtime News
on your WebpageAdd Widget >Get your members hooked!


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Why a Student Debt Strike: Join the strategy call
Tues May 5th, 1pm ET
(that's next week!)
Join Hightower in a strategy conversation with Latonya Suggs (Corinthian 15) and Ann Larson (Debt Collective).
More info:
David Burnell Smith, an attorney from Carefree, Arizona, won election to the state house in 2004. He opted for public financing --even though this antigovernment Republican insisted that he was opposed to Arizona's Clean Election law. Perhaps to show his disdain, Smith carelessly exceeded the spending limit that the law imposes. In 2005, the Clean Elections Commission investigated his case and ordered Smith out of his legislative seat, ruling that he had acquired it by cheating. Smith imperiously asserted that he was now a sitting legislator and thus immune from the commission's reach. Unfortunately for him, a string of courts, including the Arizona supreme court, disagreed with the little potentate, and he was booted.
Defiant, Smith ran again last year for the same seat, this time spurning public funding and pledging to kill the whole program. "I'll win my re-election," he spewed out. "I'll be stronger than ever. And I'm going to do what I can to rid this state of socalled clean elections…."
Alas, voters disagreed. Mr. DBS lost in the Republican primary last September, running third. He was last seen wrangling with the Clean Elections Commission to lower his fine. "They got their pound of flesh," he said. "You'd think I could negotiate something."


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This is another photo I scanned recently from a Zalewski Genealogy booklet. Back row: Mary (Gierszewski), My great-great-grandmother Anna (Lindner), my great-grandfather Joseph & Martha (Strelka) — Front row: Frances (Cybela), Helen (Stroinski) & Angeline (Pierzchalski) – Milwaukee, Summer 1925
You are here: Home / Weekly / Tell Me Thursday / Tell Me Thursday – Zalewski Family
Tell Me Thursday – Zalewski Family
11 June 2009 by Brian Zalewski
Filed Under: Tell Me Thursday Tagged With: 1920s, Milwaukee, photo, Zalewski
About Brian Zalewski
I started genealogy research about mid-1999. My grandfather had passed away in April of that year. Since then I've done a lot of research not only for myself, but for friends and other relatives. In 2006, I married the love of my life, Darcy, and welcomed the birth of our daughter, Aerissa Jean, in 2010 and our son, Xander Lee, in 2012. I can't wait to tell them stories about all of their ancestors.
Additional Resources
A Featured Post
Maps. Maps. Maps.
I started using the Google Maps system to plot some of my family's locations awhile back, but I never got around to finishing it. Recently, I plotted most of the major Milwaukee locations for my family and it's interesting to see how it looks once you know where things are. I've always had an idea, […]


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(skip this header)
Pedestrian in critical condition after crash
Top Stories in Local
US stocks slip after Greek 'no' vote; European markets sink
Top Stories in US & World
Real Estate
Alamodome renovations approved
Top Stories in Business
Joseph returns home to join Raptors
Top Stories in Sports
Shuck Shack now open
Top Stories in Food
Thrilling 'Shark Week' keeps feeding our fears
Top Stories in Entertainment
12 Texas beaches for that sand and surf glow
Top Stories in Lifestyle
Lawmakers act to improve foster care
Top Stories in Opinion
2015 Volkswagen Golf R — the current (and hot) version of the old VW Rabbit.
Top Stories in Cars
Aetna to buy Humana as health insurer landscape shifts
Top Stories in Jobs
Beautiful Hill Country ranch listed for $18.5 million
Top Stories in Homes
June 21-23 in Dover, Delaware, featuring Red Hot Chili Peppers, Tom Petty & the Heartbreakers, Vampire Weekend and more.
Owen Sweeney/Associated Press
© Copyright


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Gaza War, One Year Later: Knesset set to mark anniversary
Final vote on 2015-2016 budget set for November 19
Knesset votes to continue work on bill to force-feed hunger-striking prisoners
Gold to thank Indians for apparent shift in UN voting pattern
Classical Review: IPO
How E. Randol Schoenberg fought for The Woman in Gold
SHOW REVIEW: Cirque Du Soleil– Quidam: Menora Mivtachim Arena, Tel Aviv, July 2
Enter Bardo Pond's musical time machine
Casspi to remain in Sacramento with new two-year deal
Farmar returns to rebuilding Maccabi Tel Aviv
2015/16 soccer season to begin with fireworks
Erlich aiming to continue surprise run at Wimbledon
Analysis: Making sense of Germany's vote against Israel in the UN Human Rights Council
Iran Defense Minister warns of 'Israel's WMDs' threat to world
Shin Bet alone can't stop the slippery slope of Israeli Arabs joining ISIS
School teachers working in Israeli schools supported ISIS, Shin Bet says
Al-Quds Brigade builds watch tower directly across IDF structure on Gaza border
Haredim arrested for throwing rocks at Arab vehicles near Damascus Gate
Legendary Jewish producer Jerry Weintraub dies at 77
Clinton calls for strategy to fight boycotts of Israel
WATCH: Jewish gymnast Aly Raisman strips down for ESPN
Mazel tov: Kabbalists Kutcher and Kunis tie the knot
Philadelphia museum celebrates Jewish role in promoting gay rights
Sisi to US Jews: Israeli-Palestinian peace would make Mideast safer
Experts: Egypt in life-and-death struggle, though regime is stable
Another deadline arrives, but no deal yet over Iran's nuclear program
Hardline Revolutionary Guard set to profit from Iran nuclear deal
Iranian official: US will remain our enemy despite emerging nuclear deal
Professionals and volunteers available to counsel youth at popular entertainment spots
Barkat: Appoint permanent Hadassah Medical Organization director general to prevent collapse
Rate of women academics at universities and colleges still lags way behind US and EU
Health Scan
Opposition MKs, gov't officials, gas companies spar over controversial outline
Delek, Noble executives: We commit to developing Leviathan
Members of the public can submit reactions to gas outline until July 21
TAU collaborates with IATA on aviation technology and security
Google's Waze to start carpooling pilot program in Israel
Israel's Elbit Systems wins $150 mln Benelux contract
Apps for finding jobs are among Microsoft Venture's latest start-up
How US investors raised largest-ever real-estate bond offering in Israel
Shalom approves permits for Jordanians to work in Eilat hotels
Letters to the Editor: Measuring up
Fundamentally Freund: Bring Herzl's grandparents for reburial in Jerusalem
Exploiting anti-Semitism for the occupation's sake
Why universities need a definition of anti-Semitism
Demand BDS justify its goals
Nuclear Timeline
Taking on BDS
Boycott takes center stage
Preempting 'Goldstone II'
Israel, doctor's orders
A historian, writing in first person
A lover of life
Muslim and Jewish Jerusalemites break bread together
Cochavit Netzach Dolev takes a stand
Food for thought
Taking back control for the good of our kids
This week in Jerusalem
Art is for everyone, everywhere
Speechless theater
Extreme family fun
Opening the women's wrestling floodgates
Distancing Israeli Arab moderates instead of drawing them closer
This Islamic State madness has gone on long enough
An obsession with Israel that even trumps national self-interest
Israel-Palestine: French recipe lacks ingredients
אֲנָשִים טוֹבִים
עֵמֶק הַצְבָאִים
שֶקֶט אֲמִיתִי
חִיבּוּק שֶל אַהֲבָה
The path to aliya- Indian style
The journey of Ukraine's embattled Jews to Israel
A historic win, but no slam dunk
Roads to hell
Our island
Israel stands in the way and is an obstacle to full Muslim domination of the Middle East
Pope Francis to leave for South America in 'homecoming' trip
Episcopal Church overwhelmingly rebuffs divestment resolution
Will pope chew coca leaves in Bolivia?
Three US churches consider BDS measures against Israel in upcoming vote
NGO seeks to seize N. Korean ship to pay off $330 m. US judgment for killing of priest
Green Leadership Classroom Inaugurated at Dimona School
Eitan Levav Scenic Lookout Inaugurated in Adamit Park
Cherries in the Negev: If You Will it, It is No Dream
Healing Garden Dedicated at the Dana-Dwek Children's Hospital in Tel Aviv
Italy's National Day at EXPO 2015
The Ambassadors' Club of Israel Tours the Galilee with KKL-JNF
The International Solidarity Mission Says Goodbye in Jerusalem
Chinese Ambassador Visits the Israel-China Friendship Fores
New Bio-filters Purify Surface Runoff Water for Reuse
Future Tour Guides Discover the Negev with KKL-JNF
The Shamir Drilling Project
Graduation Ceremony for Rosh HaAyin's Volunteer Firefighters
$100 Million JNF Boruchin Israel Education Advocacy Center Opens
JNF Canada Pays Tribute to the Honourable John Baird
From Sunny Israel to Cold Canada
Walking for Water with KKL France in the Mountains of Eilat
The Negev Conference on Agricultural Research and Development
Arava Open Day: Food Security for Israel and the Entire World
Living Green in Israel: the Contest and the Winners
Addressing Climate Change Conference
An Olah Reports From Nepal
How One Oleh is Helping to Transform the Way People Drink Water
From Think Tank to Environmental Consultant
Award Winning Designer Launches Career as Wedding Dressmaker
How Natalie Edwards Built a Successful Career in Israel's Startup Industry
5 Tips to Land a Job in Israel
Starting Your Own Business in Israel: Real Life Examples
5 Strategies for Finding Your Professional Place in Israel
36 Hours in Southern Israel
Food Shopping In Israel
How Women Can Have it All in Israel
The Eisenberg's Long Journey Home
Why Retirees are Thriving in Israel
Thanks to Aliyah video
6 Frequently Asked Questions about Israeli Healthcare
Starting Over in Israel
Sam Eisenberg: From Nativ to Aliyah
WATCH: Why Olim #LiveLoveIsrael
5 Ways that Olim Kick Back
VIDEO: Are you ready for the South?
Make yourself at home… in Israel
NBN Announces Bonei Zion Prize Recipients for 2015
NefeshB'Nefesh Provides Services to Bnot Sherut
How Doctors Are Impacting Israel's South
07.07.2015 | 20 Tammuz, 5775
Weizmann scientists bring nature back to artificially selected lab mice
Health Scan: The 'memory' of starvation is in the genes
Looking young in old age
Medical Corps training, improved technologies saved lives in Gaza
German 'reneges' on optional fluoridation of drinking water
Ministry urges taking preventive measures against West Nile virus
Religious Zionists, ultra-Orthodox men free to pursue nursing degree
*Health Scan: Upgrading the stethoscope*
*Danger: Viruses may trump mankind*
MDA urges adherence to safety rules to prevent drowning
Health Ministry instructs public hospitals to return to routine
*Rx for Readers: Is alarmist ad campaign's vaccine as necessary as its sponsors say?*
Health & Science
04/27/2014 19:17
Hadassah medical workers protest in Jerusalem, February 10, 2014..
(photo credit:JERUSALEM POST)
The Histadrut-General Labor Federation said Sunday that it would postpone until after Holocaust Remembrance Day protests over the government's failure to resolve the Hadassah Medical Organization's financial straits. HMO workers at Jerusalem's two university-linked medical centers are preparing a plan, including working according to an emergency schedule and "going to the streets," said Avi Nissenkorn, the Histadrut's deputy secretary-general. "The Treasury and Health Ministry must find an immediate solution. This is the time to end the crisis and save Hadassah," he said.The union official, after meeting with representatives of the nurses' and administrative workers, said they would hold off sanctions until Tuesday if no progress is made with the government. "We will not allow HMO to be dismantled," he said. If necessary, thousands of HMO workers will demonstrate around the country, he added.Under law, sanctions and strikes will be permitted after 10 days – two weeks after the Histadrut declared a work dispute over HMO.
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Stalley is looking to take his "Intelligent Trunk Music" to new heights with his upcoming mixtape, Honest Cowboy. In his debut vlog promoting the tape, ...Read More
Stalley Searches For His Father And Explores His Inner Cowboy On New Mixtape And MMG's 'Self Made Vol. 3′
Photo Credit—Virgilio Tzaj MMG is a powerhouse in hip-hop right now, with Wale's The Gifted topping the Billboard charts and Meek Mill ...Read More
Stalley Reveals Artwork And Tracklist To 'Honest Cowboy'
Stalley is gearing up to release his new mixtape, Honest Cowboy. With a debut album on the horizon, he teases his fans with a new ...Read More
Stalley "A-Wax"
Stalley drops the first single from his forthcoming mixtape today. "A-Wax" sounds like a record off a Blaxploitation soundtrack, even though I do like the ...Read More
Stalley "Raise Your Weapons"
Stalley drops a new record called "Raise Your Weapons," the second single from his upcoming mixtape. In the wake of the Trayvon Martin trial, this ...Read More
Here's The Video For "Swangin" From Stalley Featuring Scarface
This song has so much soul. Stalley delivers the visuals for his latest single "Swangin," which features Scarface. The song is the definition of intelligent ...Read More
XXL's Sneak Preview of Wale's 'The Gifted'
On Monday (June 3), Wale hosted an intimate listening session at the movie theater level of Bryant Park Hotel, located in Midtown Manhattan. Three weeks ...Read More
Nicki Minaj & Peter Rosenberg Come Face-To-Face In The Best Instagrams This Week
This week in the world of hip-hop Instagrams, two bitterly embattled foes—Nicki Minaj and Hot 97's Peter Rosenberg—finally meet for a long-awaited interview. At ...Read More
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How did three Danes end up being tortured in Lebanon?
What a crisis has done to the people of Greece
Simon Hooper | 29 May 2013 14:12 GMT | United Kingdom
London, UK - British Muslims fear they could become "sitting targets" for far-right violence following a spate of attacks on mosques and a spike in other reported hate incidents in the week since the murder of a British soldier on a London street.
The most serious attack occurred in the east coast town of Grimsby on Sunday night when three petrol bombs were thrown at a mosque as a meeting was taking place inside.
Other attacks were reported on mosques in the southern towns of Braintree and Gillingham within hours of last Wednesday's killing.
Elsewhere, a petrol bomb was thrown at a mosque in Milton Keynes, bacon was left on the steps of a mosque in the Welsh capital Cardiff, and there were reports of vandalism at Islamic centres elsewhere.
Meanwhile, about 1,000 supporters of the far-right English Defence League rallied in central London on Monday chanting "Muslim killers off our streets" and heard Tommy Robinson, the EDL's leader, call for further demonstrations.
"They've had their Arab Spring. This is time for the English Spring," said Robinson, whose group also saw its number of Facebook followers surge from 20,000 to more than 100,000.
Spike in Islamophobia
Tell MAMA (Monitoring Anti-Muslim Attacks), a helpline set up to monitor instances of Islamophobia, said it had recorded 193 incidents since last Wednesday, compared to a usual tally of about four cases a day.
"There is a background Islamophobia that is always rumbling," Fiyaz Mughal, a director of the Faith Matters interfaith group that runs Tell MAMA, told Al Jazeera. "But if we take the latest figures, we are looking at about 40 cases a day and a ten-fold jump all of a sudden."
Salim Bhorat, a community activist in Bolton, a town in northwestern England with a large Muslim minority, said one mosque had been vandalised with spray paint, dog excrement had been thrown at another, and a woman had been attacked in the street. A car outside the mosque was also daubed with the words "Terrorist inside".
He said the killing of soldier Lee Rigby appeared to have reawakened the EDL in the area even as its influence appeared to be waning amid group infighting.
"Nothing like this has happened in Bolton for a long time, so these attacks are a direct response to Woolwich and the mass hysteria afterwards," Bhorat told Al Jazeera. "We've always had the EDL in Bolton and this has given them an excuse to rally their weary troops."
Bhorat said many local Muslims were nervous because unfounded rumours of attacks were spreading quickly via social networking and text messaging. "There has been a lot of scaremongering. People forward on messages without verifying them and that just creates a false sense of fear in the community."
'We are afraid'
Sikander Sleemy, the secretary of a mosque attacked in the Essex town of Braintree, told Al Jazeera that a smoke grenade was thrown into the building last Wednesday evening by a man armed with two knives.
"We were very fortunate because the prayer time was only 15 minutes away but there was only one person in the mosque. He was upstairs when he heard the bang and smelt the smoke and he could hear someone shouting and swearing downstairs, but he managed to escape onto the roof," he said.
These attacks and incidents are happening in areas where Muslims are the majority as well as the minority.
Fiyaz Mughal, a director of Faith Matters ,
Sleemy said Braintree's small local Muslim community had never experienced any problems before. "We do feel part and parcel of the community and my personal feeling is that this was just an isolated rage attack. But if idiots like those guys in Woolwich, if there are any more attacks like that, God forbid that there are, then we are afraid that we will become sitting targets."
Amid shock and condemnation, Justin Welby, the Archbishop of Canterbury and head of the Church of England, visited Leicester last week in a show of interfaith solidarity with Muslim leaders, praising the diverse city, where just under a fifth of residents are Muslim, as a "a shining example of how communities work together".
Suleman Nagdi, a local Muslim civic leader, told Al Jazeera he believed the city's Muslim community was well established and confident enough to face down any threats against it. "Leicester has mature relations between members of the different faith communities. We have a situation up and down the country that is concerning to us but we built those bridges many, many years ago," he said.
But Mughal questioned the assumption that Muslims in cities such as Leicester were better protected than those in areas with smaller and less established Muslim populations.
"The fact of the matter is these attacks and incidents are happening in areas where Muslims are the majority as well as the minority. In Leicester we had a cluster of incidents last year. It's quite a hot spot because there is a concentration of far-right supporters in the area around the city."
Mughal said far-right activists were increasingly using the internet not just for recruitment and dissemination purposes but also to attack Muslims online. "If you get a woman whose avatar has a hijab they will bombard here with sexual abuse to humiliate here. That is the kind of activity we are seeing."
He also cited concerns about social networking sites and new guidelines for police which have raised the threshold in terms of what is considered prosecutable as hate speech online. "Our biggest issue is constantly having thousands of tweets out there that are just promoting deeply anti-Islamic feeling. We've never had that before," he said.
Media coverage
Others raised frustrations about the mainstream media's coverage of the Woolwich attack, including the labelling of the perpetrators as "Islamic terrorists". Nick Robinson, the BBC's political editor, apologised later that day after quoting a source who described the attackers as being "of Muslim appearance".
"We are the only community, in the UK certainly, which whenever an act of terrorism has taken place, the whole community is put in the dock for the criminal activities of individuals," said Nagdi. "Acts of terrorism affect us all but it does seems odd that when it is a Muslim you always expect Muslims to condemn it. We do condemn it but there is a history of bias in the reporting mechanism that it is expected."
Sleemy also condemned the airtime given to Anjem Choudary, a radical cleric formerly of the banned al Muhajiroun group, on the BBC's flagship Newsnight programme.
"He is not the voice of Muslims. He is a very extreme fringe group, and if people are watching that and they've got very little knowledge they are building a picture of a Muslim that he has portrayed," he said.
Mughal said positive media coverage of Muslim communities would help address Islamophobia but added that tougher police action was urgently needed.
"The police have failed miserably dealing with some cases we've put through to them. They need to understand Islamophobia as a phenomenon that is deeply corrosive and they need to act quickly."
He said it was hard to predict for how long the current upsurge of anti-Muslim attacks would continue as no comparable data was available for events such as the 2005 bombings on the London transport network.
But he added: "The fact is these effects are cumulative, and this was such a significant and barbarous attack that it will certainly add to the cumulative effect over time."
Source: Al Jazeera
EDL supporters decry 'Islamification' of Britain after soldier was killed by two attackers reportedly of Muslim faith.
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"Watching people at a café or sitting by a busy street reminds me of how little time we have, and that future is always upon us".
Staffan can conjure inspiration from, what seems to be anything that is constructed with the creative minds of other people, being architecture, sneakers or listening to new music. His passion for electronic music is deeply rooted in his perfectionist´s mind. The drive towards obliterating boundaries and developing new forms and structures in sound is not only a way for him to express his creativity, but also this funtions as therapy for Staffan.
"I love to hear and watch things that are hard to recognize, whether that being a sound or an image. Working in the studio helps me clear my head".
This effacement of thought enables a fresh start, and pave ways for new ideas for Staffan.
Staffan also work alot with his brother Mathias. Together they form the dynamic duo of "Decoy Linzatti".
Edited by sonoluca on 15 Dec 2007, 17:16
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Pontiac's Rebellion
Pontiac's Rebellion began when the Ottawa Indian chief led an attack on Detroit. The conflict ended when the Indians failed to receive French aid.
Glossary Term – Person
Pontiac (1720–1769) was an American Indian leader who revolted against the British occupation of the Great Lakes regions. He led warriors from numerous tribes who joined in an attempt to drive British soldiers and settlers out of the region.


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text:
An apo(a) gene 5'-regulatory region upstream from position -208 from the translation initiation site is provided. Also included are expression constructs, vectors, translated cells, and transgenic animals comprising the regulatory region, and methods for determining expression levels and for screening for compounds which control expression.
1. A nucleotide sequence comprising the human apolipoprotein(a) gene 5'-regulatory region from nucleotide position -208 to nucleotide position -1442 of FIG. 3 nucleotides 1-1235 of SEQ ID NO: 1), operatively linked to a heterologous sequence.
2. The nucleotide sequence of claim 1 wherein said apo(a) gene 5'-regulatory region is derived from a human subject having serum Lp(a) levels of greater than 5 mg/mL.
3. The nucleotide sequence of claim 1 wherein said apo(a) gene 5' regulatory region is derived from a human subject having serum Lp(a) levels of less than 5 mg/ml.
4. The nucleotide sequence of claim 1 wherein the heterologous sequence codes for a heterologous polypeptide.
5. The nucleotide sequence of claim 4 wherein said heterologous polypeptide is an enzyme.
6. The nucleotide sequence of claim 5 wherein said enzyme is β-galactosidase.
7. The nucleotide sequence of claim 5 wherein said enzyme is luciferase.
8. The nucleotide sequence of claim 5 wherein said enzyme is chloramphenicol acetyl transferase (CAT).
9. The nucleotide sequence of claim 4 wherein said heterologous polypeptide is avidin.
10. The nucleotide sequence of claim 4 wherein said heterologous polypeptide is an immunogen.
11. A nucleotide sequence comprising a human apolipoprotein(a) gene 5'-regulatory region operatively linked to a heterologous sequence, said 5'-regulatory region comprising at least one hundred consecutive nucleotides of the regulatory region depicted in FIG. 3 from nucleotide position -208 to -1442 nucleotides 1-1235 of SEQ ID NO: 1).
12. A vector comprising the human apolipoprotein(a) gene 5'-regulatory region from nucleotide position -208 to -1442 of FIG. 3 nucleotides 1-1235 of SEQ ID NO: 1), operatively linked to a heterologous sequence.
13. The vector of claim 12 wherein the apo(a) gene 5'-regulatory region comprises an IL-6 responsive element.
14. The vector of claim 12 wherein said IL-6 responsive element comprises the sequence CTGGGA.
15. The vector of claim 12 further comprising an HNF-1α responsive element.
16. The vector of claim 12 wherein the heterologous sequence codes for a polypeptide.
17. The vector of claim 16 wherein said polypeptide is an enzyme.
18. The vector of claim 17 wherein the enzyme is luciferase.
19. An isolated nucleotide sequence comprising at least thirty consecutive nucleotides (1) from the human apolipoprotein(a) gene 5'-regulatory region depicted in FIG. 3 from nucleotide position -208 to -1442 nucleotides 1-1235 of SEQ ID NO: 1) or (2) from the complement of said 5'-regulatory region.
20. A nucleotide sequence comprising at least fifteen nucleotides capable of forming a triplex DNA complex with a double stranded DNA comprising the human apolipoprotein(a) gene 5'-regulatory region depicted in FIG. 3 from nucleotide position -90 to -1442 nucleotides 1-1353 of SEQ ID NO: 1).
21. The nucleotide sequence of claim 20 wherein said triplex DNA complex is formed with an IL-6 responsive element.
22. The nucleotide sequence of claim 20 wherein said triplex DNA complex is formed at a site comprising nucleotide -913 (nucleotide 529 of SEQ ID NO:1).
23. The nucleotide sequence of claim 20 wherein said triplex DNA complex is formed at a site comprising the sequence TTTTA.
24. A transfected mammalian cell containing the nucleotide sequence of claim 5, or progeny of said transfected cell.
25. The nucleotide sequence of claim 11 wherein said apolipoprotein(a) gene 5'-regulatory region comprises an IL-6 responsive element.
26. The nucleotide sequence of claim 25 wherein said IL-6 responsive element comprises the sequence CTGGGA.
27. The nucleotide sequence of claim 11 wherein said apolipoprotein(a) gene 5'-regulatory region comprises a HNF-1α responsive element.
28. The nucleotide sequence of claim 11 wherein said apolipoprotein (a) gene 5'-regulatory region comprises an CEBP responsive element.
29. The nucleotide sequence of claim 28 wherein said CEBP responsive element comprises the sequence GCAAT.
30. The nucleotide sequence of claim 11 wherein said apolipoprotein(a) gene 5'-regulatory region comprises a LF-AI responsive element.
31. The nucleotide sequence of claim 30, wherein said LF-AI responsive element comprises the nucleotide sequence CTGGGATTACAGAGTTGAGCCA (nucleotides 303-324 of SEQ ID NO: 1).
32. The nucleotide sequence of claim 30, wherein said LF-AI responsive element comprises the sequence TGAGCCA.
33. The nucleotide sequence of claim 11 wherein said apolipoprotein(a) gene 5'-regulatory region comprises an IL-6RC responsive element.
34. The nucleotide sequence of claim 33, wherein said IL-6RC responsive element comprises the sequence CTTTCACCA.
35. The nucleotide sequence of claim 11 wherein the heterologous sequence codes for a heterologous polypeptide.
36. A vector comprising a human apolipoprotein (a) gene 5'-regulatory region operatively linked to a heterologous sequence, said 5'-regulatory region comprising at least one hundred consecutive nucleotides of the regulatory region depicted in FIG. 3 from nucleotide position -208 to -1442 nucleotides 1-1235 of SEQ ID NO: 1).
This application is a continuation application of U.S. application Ser. No. 07/991,849, filed Dec. 15, 1992, abandoned, which is incorporated herein by reference in its entirety,
The field of the invention pertains to 5' promoter and regulatory element sequences which control expression of apolipoprotein(a); and to constructs and vectors comprising apolipoprotein(a) regulatory sequences operatively linked to heterologous sequences.
Lipoprotein(a) Lp(a)! is a variant lipoprotein which has been shown to play a role in development of atherosclerosis. Because atherosclerosis is the leading cause of death in the United States, interest in the structure and function of Lp(a) has greatly increased in recent years. Epidemiological studies have shown a population-attributable risk of 28% for suffering myocardial infarction before sixty years of age in those men having an Lp(a) concentration in the top quartile of the population distribution (Rhoads et al., JAMA (1986) 256:2540-4). A study conducted in England found that nearly all instances of familial premature coronary heart disease occurring in the absence of classical hyperlipidemias were associated with high Lp(a) levels (Durrington et al., Lancet 1988:1070-3).
Lp(a) is known to constitute a cholesteryl-ester rich low density lipoprotein (LDL)-like particle which closely resembles LDL in content of cholesterol, phospholipid, and apolipoprotein B-100 (apoB). ApoB is the ligand for the LDL receptor. Binding of ApoB to the LDL receptor provides an uptake mechanism for moving LDL into cells, where it provides cholesterol needed for membrane and steroid synthesis. Like LDL, Lp(a) consists of a core of cholesteryl esters and triglycerides, and a surface layer of phospholipids and free cholesterol. In both LDL and Lp(a), the single hydrophobic molecule of apoB is arranged primarily beneath or near the surface of the particle.
Lp(a) is distinguished from LDL by the presence of an additional apolipoprotein known as apo(a), a glycoprotein which is bound to apoB through a disulfide linkage. Unlike the predominantly hydrophobic apoB, apo(a) is a hydrophilic glycoprotein. The disulfide linkage between apoB and apo(a) occurs on a hydrophilic loop of apoB which extends into the aqueous medium surrounding the particle.
Recent studies have provided sequence information about the apo(a) protein and the coding region of the apo(a) gene. Apo(a) comprises a variable number of kringle domains linked to a C-terminal protease domain. The protein and nucleotide sequences of apo(a) are strikingly homologous to the sequences of certain domains of the clotting factor protein plasminogen. Mature plasminogen consists of five distinct kringle domains, numbered 1-5 starting from the N-terminus, and a C-terminal protease domain related to the serine protease trypsin. The five kringles of plasminogen share about 50% sequence identity with one another. They bind plasminogen to its fibrin substrate and to specific inhibitory proteins.
The N-terminal kringles of apo(a) comprise repeating domains most closely related to kringle-4 of plasminogen. The number of copies of the kringle-4-like domain varies dramatically among human alleles. Within the general population, the number of repeated kringle-4 domains appears to range from 15 to 40. This genetic diversity is reflected in the molecular weight variation of mature apo(a), which ranges from 300 to 800 kD (Koschinsky et al., Biochem. (1990) 29: 640-4). In one sequenced cDNA clone, 22 tandem exact repeats and 15 modified repeats of kringle-4 were found. These copies exhibit from 75% to 85% nucleotide identity to plasminogen kringle-4.
Moreover, an even greater sequence identity was observed between kringle-5 of plasminogen and a single copy of an apo(a) kringle, located C-terminal to the kringle-4-like repeated domains of apo(a). The kringle-5-like domain exhibits 91% nucleotide sequence identity with kringle-5 of plasminogen.
Immediately C-terminal to the kringle-5-like domain is a protease domain which exhibits 94% nucleotide and 88% amino acid sequence identity with the protease domain of plasminogen.
Even more pronounced homology is found at the 5' end of the transcribed gene message. Plasminogen and apo(a) contain 5'-untranslated sequences which share 98% sequence homology, followed by a signal peptide sequence which is 100% identical.
Covalent linkage of the apo(a) moiety to apoB is believed to inhibit uptake of Lp(a) by the LDL receptor. Such uptake is the primary means for clearing LDL from plasma. The diminished cellular uptake observed for Lp(a) suggests that regulation of Lp(a) serum levels may depend more upon control of the rate of Lp(a) synthesis than upon the rate of metabolism.
Although elevated Lp(a) levels pose a significantly increased risk of premature coronary heart disease, no practicable method for lowering the serum concentration of Lp(a) is presently known. Unlike LDL levels, Lp(a) serum concentration appears to be insensitive to dietary or exercise regimens. Instead, the Lp(a) level apparently is genetically determined, and remains relatively constant throughout the lifetime of a given individual. Elevated Lp(a) levels appear to be inherited as a dominant trait under single-locus control.
Furthermore, with the exception of nicotinic acid, all drugs tested to date have failed to reduce Lp(a) concentrations. Even HMG-CoA reductase inhibitors, which dramatically reduce LDL concentration by inhibiting cholesterol synthesis, fail to lower the concentration of Lp(a).
Consequently a need exists for improved methods and compositions for reducing the serum concentration of Lp(a). In particular, lower Lp(a) levels might be obtained by limiting the expression of its unique component, apo(a). However, at present very little is known about the physiological or hormonal control of apo(a) expression.
One approach to deciphering the regulation of apo(a) expression is to isolate the 5' genomic regulatory elements. The upstream regulatory sequences of numerous mammalian genes have been shown to contain regulatory "elements", i.e., relatively short nucleotide sequences which either enhance or inhibit transcription of the proximal coding region. Most regulatory elements are believed to function by binding particular regulatory proteins which can selectively associate with a specific DNA sequence. These DNA-binding regulatory proteins may provide for tissue-specific developmentally and hormonally regulated gene expression.
Certain DNA-binding regulatory proteins are known to be receptors for particular hormones, such as glucocorticoid, estrogen, and thyroid hormones. Each hormone receptor targets a distinct DNA site or response element. In general, binding of the hormone to the receptor protein alters the affinity of the receptor protein for its cognate DNA response element. In the case of lipid-soluble hormone receptors, binding of the hormone-receptor complex to its DNA response element stimulates transcription, i.e., exhibits positive regulation.
Other examples of positive regulatory elements are provided by transcription enhancing factors activated by hormones which bind to cell surface receptors, such as interferon α. Binding of interferon α to its cell surface receptor leads to the association of three cytoplasmic proteins, each of which is inactive by itself, to form the active interferon α-stimulated factor. The active factor is translocated to the cell nucleus and in turn stimulates transcription of those genes having a cognate DNA binding site.
In addition to these examples of positive or up regulation of transcription, regulatory elements which confer suppression of transcription also have been identified in some mammalian genes. For example, a suppressive sterol regulatory element (SRE) has been found in the upstream region of the LDL receptor gene (U.S. Pat. No. 4,935,363 to Brown et al.). Frequently both positive and negative regulatory elements occur within the same upstream regulatory region, as has been reported for the gene for apoA-I. ApoA-I is a principal apoprotein component of high density lipoproteins (HDL) (Papazafiri et al., J Biol. Chem. (1991) 266:5790-7).
If a comparable understanding of the regulatory elements responsible for positive and negative control of transcription of apo(a) could be obtained, a rational search for therapeutics might be conducted. However, although the coding regions of the apo(a) gene have been cloned and sequenced, the regulatory regions thus far have not been isolated or characterized. At least in part, the failure to identify clones containing the apo(a) 5'-regulatory region may reflect difficulties arising from the unusually high degree of homology between apo(a) and plasminogen. The extreme homology between the 5' portions of the plasminogen and apo(a) genes is particularly significant because the 5' transcribed region is immediately adjacent to the upstream regulatory region. Therefore, probes directed to the 5'-transcribed region would be especially useful for screening a genomic library to identify the apo(a) regulatory sequences. The extensive identity between apo(a) and plasminogen nucleotide sequences indicates that such probes would be expected to cross-react with the plasminogen gene even under stringent conditions of hybridization.
To further complicate identification of the apo(a) regulatory region, recent studies by Ichinose, Biochemistry (1992) 31:3113, and Ichinose et al., Proc. Natl. Acad. Sci. USA (1991) 88:115, have demonstrated that plasminogen and apo(a) are members of a multiple gene family. At least two distinct genomic clones coding for apo(a) were reported, although one or both could be pseudogenes. Furthermore, three other plasminogen-related genes (PRGs) or pseudogenes having high homology to parts of the plasminogen and apo(a) coding sequences also were observed. Finally, hepatocyte growth factor has been found to constitute yet another member of the plasminogen-apo(a) gene family (Miyazawa et al., Biochemistry (1991) 30:9170-6).
Not surprisingly in view of the presence of this extensive family of related genes, a cDNA probe comprising a plasminogen kringle-4 sequence identified a large number of genomic fragments on a Southern blot (Ichinose, supra, pp. 3113-4and FIG. 1 therein).
Despite these anticipated difficulties in identifying genomic regulatory regions of apo(a), the search for such regulatory regions holds great promise for devising physiological or pharmacological interventions in the level of expression of apo(a). Such intervention would be extremely useful in preventing premature coronary heart disease.
FIG. 1 shows homologous regions from the 5'ends of members of the apolipoprotein(a)/plasminogen gene family. On the right is seen a blot hybridization of human genomic DNA digested with EcoRI, electrophoresed, transferred and probed with PCR-A, a 483 bp fragment from the 5' end of the plasminogen gene. The hybridizing fragments are 4.8, 3.6, 3.1 and 1.2 kb in length. Local regions of DNA sequence identity to the 4.8 kb A fragment are shown. The fragments are shown aligned to the ATG translation initiation codon. Exon one begins at the transcription start site and ends at a splice donor sequence shared by all four fragments. The location of repetitious "Alu" sequences and their orientation is shown. The D fragment size class consists of three or more non-allelic species containing over 95% sequence identity throughout their length, one of which corresponds to the plasminogen gene.
FIG. 2 is a map of the transcription start site (arrow) of apolipoprotein (a). An antisense primer complementary to nucleotides 60-72 bp downstream of the ATG of human apo(a) cDNA (McLean et al., Nature(1987) 330:132-137) was annealed to 20μg of human liver poly (A+) RNA and extended with reverse transcriptase as described in Ausabel et al., Current Protocols in Molecular Biology (1991) pp. 4.8.1-4.8.3. A DNA sequence ladder is included as a size marker.
FIG. 3 (SEQ. ID NO: 1) shows the sequence characteristics of the 5' region of the human apolipoprotein(a) gene. Partial sequence is presented of the EcoRl "A" fragment; the complete 4798 bp sequence is available from GenBank. The first nucleotide of the translation initiation codon is numbered one. Sequences resembling CAAT, TATAA boxes and other consensus sequences for transcription regulatory elements are boxed, and exon one is underlined. The "A2" allele isolated from individual with low plasma and reporter gene expression of apo(a) (see FIG. 4 and text) has two sequence differences with the above: an insert of TTTTA indicated by v and a G to A substitution indicated by *.
FIG. 4 is a graph showing transcription activity of constructs comprising homologous genomic fragments from the apo(a) 5' region. 5' flanking regions of the genomic fragments were ligated just upstream to the coding region of the luciferase gene, transfected into HepG2 cells and assayed for luciferase activity. Values are the result of 3-5 independent experiments and are presented relative to the activity of the D1 fragment, "Con" is the luciferase plasmid pGENELIGHT-2 without insert. "A1" is the apo(a) genomic fragment derived from an individual with 30 mg/dl plasma apo(a). "A2" is the corresponding fragment from an individual with <1 mg/di plasma apo(a). "D1", "D2" and "D3" derive from three distinct plasminogen-like 1.2 kb EcoRI genomic fragments isolated from a single individual.
FIG. 5 is a graph showing transcription activity in the presence and absence of added IL-6 in HepG2 hepotocarcinoma cells transfected with plasmid A1 containing the apo(a) gene 5' flanking region from -1442 to -12 inserted into a luciferase reporter construct.
The present invention provides 5'-promoter and regulatory element sequences which control expression of apolipoprotein(a); constructs and vectors comprising apolipoprotein(a) regulatory sequences operatively linked to coding sequences for a heterologous protein, or for an apolipoprotein(a) antisense sequence; oligonucleotide sequences which form triplex complexes with apolipoprotein(a) regulatory sequences; and transgenic animals comprising a heterogenous apolipoprotein(a) regulatory sequence operatively linked to a heterologous protein or to a heterogenous apolipoprotein(a) coding sequence. The invention also provides methods for regulating the expression of apolipoprotein(a); methods for treating elevated serum levels of Lp(a); and methods of screening for compounds capable of modulating the expression of apolipoprotein(a).
As applied to an apo(a) regulatory region, "isolated" means an identified nucleotide sequence which is purified to remove regulatory regions from other members of the plasminogen gene family, and which is covalently linked to no more than 10 kb of sequence immediately upstream from the transcription start site of the apo(a) gene. In particular embodiments, isolated sequences may comprise no more than 7, 5, 3, 2, or 1.45 kb of sequence immediately upstream from the transcription start site, as indicated.
As applied to a nucleotide construct comprising an apo(a) regulatory region said to be operatively linked to a coding sequence, "operatively linked" means that the regulatory region is covalently linked 5' to a transcription initiation site, RNA coding sequence, and transcription stop site, whereby said RNA coding sequence is transcribed when the construct is introduced into a suitable cell, such as an hepatocyte or an hepatocarcinoma cell.
As applied to an apo(a) regulatory region operatively linked to a heterologous sequence, "heterologous sequence" means any sequence other than a naturally occurring human genomic DNA sequence coding for apolipoprotein(a). A heterologous sequence includes in one embodiment a sequence coding for a reporter protein, such as luciferase or β-galactosidase. In another embodiment, a heterologous sequence includes a transcribed but untranslated sequence which is antisense to all or a portion of the "sense" sequence coding for human apo(a). In another embodiment, a heterologous sequence includes a sequence coding for human apolipoprotein(a).
As applied to a nucleotide sequence comprising a human apo(a) gene 5'-regulating region greater than 208 bp upstream from the translation start site, the phrase "greater than 208 bp upstream from the translation start site" refers to the portion of the apo(a) regulating region more than 208 bp upstream from the wild-type apo(a) translation start site, whether or not said nucleotide sequence contains the wild-type apo(a) translation start site. The nucleotide sequence optionally may comprise all or part of the apo(a) 5'-regulatory region from -208 to -1 bp.
The present invention provides the 5' regulatory region of human apo(a). FIG. 3 (SEQ. ID No. 1)gives the sequence of the 5' regulatory region from -1442 through the ATG translation initiation codon. The sequence from -208 through the ATG has been reported previously, (Ichinose, Biochem. (1992) 31:3113-3118) as has the coding sequence for apo(a).
Analysis of the regulatory sequence reveals several significant features. First, at least nine sequences corresponding to known regulatory elements appear within the -208 to -1442 regulatory region. Four of these have the IL-6 response element sequence CTGGGA (Hattori et al., Proc. Natl. Acad. Sci. USA (1990) 87:2364). A fifth example occurs beginning at position -55, although the presence of this element was not noted by Ichinose. IL-6 has been shown to promote the synthesis of certain protective plasma proteins (α-cells) "acute-phase proteins") in the liver in response to infections. However, apo(a) was not previously known to be inducible by IL-6.
Another apparent response element which appears in the -208 to -1442 region is the CEBP consensus sequence 1 GCAAT, which occurs beginning at -253 and again beginning at -911. This latter occurrence is in close proximity to an single nucleotide change at -913 occurring on an allelic variant which has been shown to express apo(a) at low levels. This observation supports the inference that this CEBP response element is active in regulatory expression of apo(a).
The reverse complementary sequence to the CEBP response element (denoted CEBP-RC in FIG. 4) occurs at -195. The presence of this sequence was not noted by Ichinose.
A third apparent response element occurring in the -208 to -1442 region is for LF-AI, consisting of the heptanucleotide TGAGCCA beginning at position -1225. Of some interest is the occurrence of this sequence only 15 bp 3' to the beginning of an IL-6 response element. The 22 nucleotide sequence (contained in SEQ. ID NO: 1) containing both response elements.
provides a unique target for oligonucleotide therapeutics intended to regulate the expression of apo (a).
An alternate form of the LF-AI sequence, TGAACC, occurs beginning at nucleotide -147. Here also, the presence of this response element was not noted by Ichinose.
Another striking sequence motif which occurs beginning at -1411: the pentanucleotide TTTTA is repeated eight times in tandem. Notably, the allelic variant which expresses low levels of apo(a) has a ninth repeat of the motif. The extra tandem repeat apparently coincides with the presence of the mutation at position -914 observed in the low-expression allele given in the A2 construct.
An embodiment of the present invention provides an apo(a) 5'-regulatory region comprising a region greater than 208 pb upstream of the human apo(a) translation start site, operatively linked to a sequence which is antisense to a transcribed sequence of an apo(a) gene. The antisense sequence is sufficiently long (greater than 15 bp) to bond selectively to mRNA transcribed from the wild-type apo(a) gene. Preferably such antisense sequence corresponds to a region at the 5'-end of the translated mRNA sequence; however, due to the high level of homology with plasminogen at the leader sequence, the preferred antisense target will be outside the leader sequence domain. Preferred antisense targets are regions of maximal sequence difference between apo(a) and plasminogen.
The antisense constructs may be inserted into hepatotrophic vectors, or may be used without vector sequences. The constructs are used to transfect liver cells. Transfected cells transcribe both the natural apo(a) mRNA and the antisense oligonucleotide. The two sequences duplex, thereby inhibiting expression of apo(a) protein.
The antisense construct may be targeted to hepotocytes using a hepototrophic virus vector, such as hepatitis virus. Alternatively, the antisense construct may be incorporated within or linked to a liposome having a protein component which is targeted to a hepatocellular receptor, such as apoB and the LDL receptor.
The antisense constructs and vectors may be used to transfect cells in vitro or administered to a person to downregulate apo(a) expression.
Triplex oligonucleotides provide another means for downregulating apo(a) expression. Rules for devising a triplex-forming oligonucleotide have been devised; see Chastain et al, Nucleic Acids Res. (1991) 20:315-18; Orson et al, id. (1991) 19:3435-41; Beal et al, Science (1991) 251:1360-63; Maher et al, Science (1989) 245:725-30. In general, the triplex-forming oligonucleotide has the sequence correspondence:
oligo 3'--T G G T
duplex 5'--A G C T
duplex 3'--T C G A
Thus for any given sequence in the apo(a) regulatory region upstream from position -208, a triplex forming sequence may be devised.
Triplex-forming oligonucleotides preferably are at least 20, more prefereably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp.
The triplex-forming DNA may have a variant structure to limit degradation, such as a 3' amine termination site.
Targets preferably include at least one region containing a regulatory element and adjacent sequence. A particular target of interest is the region comprising IL-6 and LF-AI response elements at -918 to -940. Another target includes position -913 and the proximal CEBP regulatory element.
Electrophoresis, blot hybridization and other standard molecular biology procedures essentially followed the protocols of Sambrook, Fritsch and Maniatis. Genomic libraries in bacteriophage lambda (LAMBDA-ZAP 11 and GIGAPACK GOLD 11; Stratagene) were constructed from human genomic DNA digested to completion with Eco RI and screened with the probe pcr A (see below). In some cases, the Eco RI digested DNA was size-selected by fractionation by 1% agarose gel electrophoresis. Positive clones were isolated as pBLUESCRIPT plasmids by the in vivo excision procedures detailed by the manufacturer, and sequenced to completion by the Sanger di-deoxy method using the SEQUENASE kit (United States Biochemical Corporation) and oligonucleotide primers derived either from vector sequences, or from previous sequence analysis of the cloned inserts. In some cases, nested deletion sets were produced by the exo III mung bean system (Stratagene).
Primer sequences (pcr 6) (SEQ. ID NO:2) GCCTGTTGGAAAGCTTG and (pcr 7) (SEQ. ID NO:3) AGTAGAAGAACCACTTC were used to generate the genomic DNA 483 by probe designated PCR-A. ACCACATGGCTTTGC (SEQ. ID NO:2) the reverse complement of sequence in the first kringle region of apo(a) mRNA, was used to determine the mRNA start site by reverse transcriptase extension. Human liver poly (A+) RNA was isolated by the guanidium isothiocyanate procedure.
In order to assay promoter activity, a series of reporter gene constructs was synthesized (pLuc A-D), comprising the 5' flanking regions of the apo(a) gene and its homologues inserted into the polylinker region of pGENELIGHT-2 (Promega Inc., Wisc.), a promoterless vector containing the firefly luciferase gene. The genomic inserts were isolated from modified pBLUESCRIPT plasmids of the original clones from which genomic fragments 3' to the desired flanking regions had been removed by restriction enzyme digestion. The flanking region inserts were isolated from these modified plasmids by digestion with restriction enzymes appropriate for directional ligation into the pGENELIGHT-2 poly-linker. The inserts contained 1.4 kb of DNA upstream of a Bal I site located 12 nucleotides before the translation initiation ATG for the A1, A2 and B fragments, 1.07 kb upstream of this site in the D fragments, and 3.0 kb upstream of a Hinc II site at -54 bp for the C fragment.
The insert for plasmid pLucA1 was isolated using an intermediate plasmid constructed from a Cla1-Ball digest of the original Bluescript plasmid containing the 4.8 kb A1 insert. The resulting Bluescript vector containing 1.4 kb of 5' flanking region of the 4.8 kb insert terminating at a Bal1 site -12 bp from the ATG, was gel-isolated, the Cla1 cohesive end blunted by Klenow extension, and self-ligated. Digestion of this plasmid with Sac1-Xho1 produced an insert containing 1.4 kb of 5' flanking region of the 4.8 kb which was cloned into Sac1-Xho1-digested GeneLight 2 plasmid. Plasmid pLucA2 was constructed from an intermediate plasmid obtained by Bal1-Smal digestion of the Bluescript plasmid containing the 4.8 kb A2 insert. The resulting fragment containing the vector and 1.4 kb 5'flanking region of the A2 insert, terminating at the Bal2 site terminating at -12 bp from the ATG, was blunt end ligated and the 1.4 kb fragment excised by Kpn1-Spe1 digestion and cloned into the Kpn1-Nhe1-digested gene-light 2 plasmid. Plasmid pLucB was constructed with an insert derived from Spe1-Bal1 digestion of the Bluescript plasmid containing the 3.6 kp B insert. The resulting vector insert fragment containing 1.4 kp 5' flanking sequence of clone B, terminating at the Ba1 site -12 from the ATG was gel-isolated, the Spe1 cohesive end blunted with Klenow enzyme and the construct self-ligated. A fragment containing 1.4 kb of 5' flanking sequence was excised from this plasmid by Xbal-EcoR V digestion and ligated into Sma1-Nhe1 sites of the GeneLight 2 plasmid polylinker. Plasmid pLuc C was constructed from an intermediate plasmid produced by HincII digestion of the Bluescript plasmid containing the 3.2 kb C clone. This excises a 200 bp fragment leaving a vector insert fragment containing 3 kb of 5' flanking region of the 3.2 clone terminating at a HincII site -54 bp from the ATG. After religation of the plasmid, the 3 kb 5' flanking region was excised by Sac1-Xho1 digestion, and separated from the 3 kb Bluescript vector by gel-isolation, following digestion with Sca1, which cuts the vector into 1 kb and 1.8 kb fragments, and cloned into the Sac1-Xho1-digested GeneLight 2 plasmid. pLuc D1 and D2 were constructed from intermediate plasmids produced by Ba1-Sma1 digestion of the Bluescript plasmids containing these inserts followed by gel-isolation and self-ligation of the resulting vector-insert fragment containing approximately 1 kb of 5'flanking sequence terminating at the Ba1 site at -12 bp from the ATG. The 5' flanking insert was excised by Sac1-Kpn1digestion and cloned into Sac1-Kpn1 digested GeneLight 2 vector. pLucD3 was constructed from an intermediate plasmid produced by digestion of the Bluescript D3 clone with Hinc2, followed by gel-isolation of the 4 kb vector insert fragment and self-ligation resulting in a fragment containing 1 kb of 5' flanking region of D3 terminating at the Hinc2 site at -54 bp from the ATG. This fragment was excised by Xho1-Sac1digestion and cloned into these sites in the GeneLight2 vector.
Plasmids for transfection were purified by two cesium chloride density gradient ultracentrifugations or by QIAGEN columns (Qiagen Inc., Calif.). For transient transfections with luciferase construct plasmids, HepG2 cells were seeded at 0.75�108 cells per 6 cm dish and maintained in DMEM (Gibco-BRL) medium supplemented with 10% FCS. Transfections were performed when the cells attained 40-60% confluence. For each dish, 10 μg of test plasmid DNA and 5 μg of pSV-β-galactosidase control plasmid (Promega Inc., Wisc.) were mixed with 1.5 ml of Opti-Mem medium (Gibco-BRL) in a polystyrene tube and 1.5 ml of Opti-Mem containing 30 μg of Lipofectin (Gibco-BRL) was added. Complexes were allowed to form for 30 mins. at room temperature and the lipofectin-DNA mix was added to the dishes after washing the monolayers three times in Pucks Saline A (Gibco-BRL). After 16 hours the medium was replaced by 5 ml of DMEM containing 10% FCS, and incubation continued for 48 hours before assay of cellular luciferase and β-galactosidase activity.
Monolayers were washed five times with ice-cold phosphate-buffered saline (PBS), scraped in 1 ml PBS and pelleted by centrifugation at 4� C. for 10 seconds in a microfuge. The pellet was resuspended in 100 μl of 0.1M KH2 PO4 buffer, pH7.8, 1mM DTT, and lysed by three freeze-thaw cycles with vigorous vortexing between each cycle. Lysates were stored in aliquots at -70� C. if not assayed immediately. Luciferase activity was measured by mixing 100 μl of luciferase substrate solution (Promega Inc.) with 20 μl of cell lysate and a 0-10 second integral read in a Monolight 2001 luminometer (Analytical Luminescence Laboratories). β-Galactosidase activity was measured after dilution of the lysates one in one hundred in KPO4 buffer. Diluted lysate (20 μl) was incubated for one hour at room temperature with 200 μI of 10 μg/ml AMPGD chemiluminescent β-galactosidase substrate (Tropix Inc., Mass.) in 0.1M KPO4, 1mM MgCl2 pH 7.8. Light emission was initiated by addition of 300 μl of 1M NaOH containing 10% volume to volume Emerald enhancer (Tropix Inc., Mass.), and a 0-10 second integral read immediately in a luminometer. Luciferase activities were normalized to β-galactosidase activity for each dish. Within each experiment luciferase activity was determined in duplicate dishes, and each plasmid was tested in 3-5 separate experiments; therefore results are expressed as the mean of 6-10 values.
Transgenic mice are produced by injecting fertilized eggs with a construct containing human apo(a) cDNA (isoform M.sub.τ= 550,000 Da; Kochinsky et al., Biochem. (1991) 30:5044-51) under the regulating control of the human apo(a) regulatory region as given in FIG. 3 (SEQ. ID NO:1. The construct contains 1.44 kb of human apo(a) regulating region upstream of the translation start site, the apo(a) secretion signal sequence. 17 tandem copies of a 342-base pair kringle-4-like domain, a kringle -5 domain, and a protease domain. The 3'-untranslated region of apo(a) is fused to an SV40 early region polyadenylation signal.
This construct is microinjected into fertilized hybrid mouse eggs by the procedure of Brinster et al., Proc. Natl. Acad. Sci. USA (1985) 82:4438-42. The founder transgenic mouse is derived from C57BL/6�SJL F2 eggs and is mated to a C57BL/6�SJL F1 to establish a line. The line is maintained by continued crossing to C57/BL6�SJL hybrids. Control mice are non-transgenic litter mates.
After 6 weeks of age, transgenic mice and non-transgenic siblings are switched from a normal mouse chow diet (Techlad, Madison, Wisc.; 4% mouse/rat chow) to an atherogenic diet containing 1.25% cholesterol, 7.5% saturated fat as cocoa butter, 7.5% casein and 0.5% sodium cholate for approximately 31/2 months. At that time, plasma apo(a) is determined by ELISA (GeneScreen, Dallas, Tex.) and total cholesterol by the cholesterol oxidase method. For each animal, four 10 μm sections separated by 50 μm are quantitated. The first and most proximal section to the heart is taken 80 μm distal to the point where the aorta becomes rounded. The area of Oil Red O staining in each section is determined with a calibrated eyepiece, and the mean lesion area per section per animal is calculated for each individual and group of animals. The coded slides are examined blind in two separate analyses by the same examiner.
Lipid and apo(a) accumulation in the aorta of transgenic mice is evaluated by microscopic examination. Low (50�) (a,c) and high (150�) magnification views of adjacent sections of proximal aorta from an apo(a) transgenic mouse fed an atherosclerotic diet are stained for lipid and apo(a).
The heart and attached aorta are fixed in 10% formalin and 10 μm sections prepared. Lipid sections are stained with Oil Red O and hematoxylin, and counterstained with light green. For apo(a) immunostaining, sections are pre-incubated with 1% rabbit serum for one hour, and subsequently with 1:1000 sheep anti-human Lp(a) antibody absorbed against plasminogen (Immuno, Vienna). This antibody has no appreciable cross reactivity against mouse plasminogen as determined by Western blotting and by immunostaining of control and transgenic mouse liver sections. Bound primary antibody is detected by incubation with biotinylated rabbit anti-sheep IgG at 1:200 followed by quenching of endogenous peroxidase activity with 0.6% H2 O2, incubation with avidin biotinylated peroxidase complex, and color production with 3-amino-9-ethyl carbazole following the vendors protocols (Vector Laboratories, Burlingame, Calif.). Control slides are prepared with non-immune sheep serum in place of the primary antibody.
Co-localized apo B and apo(a) immunostaining of transgenic aorta is determined by microscopic examination of adjacent sections of aorta stained for apo(a) or apo B.
The proportions of free and lipid-associated apo(a) from the plasma of transgenic mice on the atherogenic diet is determined. 500 μl of pooled plasma from the three apo(a) transgenic mice on the atherogenic diet was adjusted to d =1.215 g/ml with KBr and centrifuged. After adjusting to equal volumes, aliquots of the top and bottom fractions are electrophoresed on a 6% SDS polyacrylamide gel under reducing conditions, transferred to nitrocellulose, and probed with sheep anti-human apo(a) (Immuno, Vienna) at 1:400, followed with peroxidase-conjugated anti-goat IgG at 1:400 and color development with 4-chloro-1-naphthol. The ratio of apo(a) in dilutions of the bottom (lipid free) and top fractions is used to estimate the proporation of the plasma apo(a) is lipid associated.
cDNA sequence analysis had shown that the 5' ends of human apo(a) and plasminogen mRNA are nearly identical. Initial hybridization of human genomic DNA Southern blots with fragments from the 5' region of the human apo(a) cDNA indicated the existence of several distinct genomic homologues of this region. A 483 bp probe (designated pcr A) covering this region was synthesized by PCR from genomic DNA, based upon plasminogen gene sequence of Peterson et al. (Peterson, T. E., Martzen, M. R., Ichinose, A. and Davie, E. W. (1990) J. Biol. Chem. 265, 1604-6111). When hybridized to an EcoRI digest of human DNA at high stringency, the pcr A probe detects four fragments designated A(4.8 kb), B(3.6 kb), C(3.1 kb), and D(1.2 kb) (FIG. 1 insert). The intensity of the D fragment indicated that it might contain two or more copies. Several clones corresponding to each of these hybridizing genomic fragments were isolated from EcoRI complete digest lambda libraries and sequenced to completion. Sequence analysis confirmed the existence of more than four extremely homologous fragments of the genome, since 3 variants of the 1.2 kb fragment were isolated from one individual (FIG. 1). Comparing the sequence of the cloned genomic fragments to the published cDNA sequence of human apo(a) and plasminogen (Sambrook, J., FFritsch, E. and Maniatis, T. Molecular Cloning, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, Malinowski, D. P., Sadler, J. E., and Davie, E. W. (1984) Biochemistry 23, 4243-4250, Forsgrent, M., Raden B., Isnaelsson, M., Larsson, K., and Heden, L. P. (1987) FEBS Lett. 213, 254-260), we found that all four fragments contain a region of over 95% sequence identity which encompasses the translation initiation codon and first exon of these genes, and contains the identical sequence CAG/GTA at the first splice donor site (The first exon of both the human plasminogen and apo(a) genes ends at an identical location 49 nucleotides following the ATG, and codes for the first 16 amino acids of the signal peptide sequence.) The 4798 bp of our A fragment sequence contain an exact match with the 400 bp sequence of apo(a) I given by Ichinose (Ichinose, A. (1992) Biochemistry 31, 3113-3118), and but one mismatch (at -21 bp before the ATG) with the overlapping region of apo(a) mRNA reported by McLean et al. (McLean, J., Tomlinson, J., Kuang. W., Eaton, D., Chen, E., Fless, G., Scanu, A., Lawn, R. (1987) Nature 330, 132-137). The assignment of the A fragment to the bona fide apo(a) is consistent with the recovery of overlapping lambda genomic clones which contain the A fragment sequence with clones encoding kringle regions of apo(a). The transcription start site of apo(a) mRNA was determined by primer extension with reverse transcriptase (FIG. 2). The major start site appears to occur at 141 bases before the translation start ATG. This is twenty bases farther downstream than the transcription start site reported for human plasminogen mRNA by Malgaretti et al. (Malgaretti, N., Bruno, L., Pontoglio, N., Candiani, G., Meroni, G., Ottolenghi, S., and Taramelli, R. (1990) Biochem. Biophys. Res. Comm. 173, 1013-1018). The sequence of the 5' region of the human apo(a) gene is given in FIG. 3 (SEQ. ID NO:1).
To begin analysis of elements that control transcription of the apo(a) gene,and to determine which of the other promoter fragments are functional, the cloned genomic fragments were inserted into luciferase reporter plasmids. Genomic DNA from the 5'Eco RI site of each fragment to a point in the 5'untranslated region was inserted into the GeneLight-2 plasmid (Promega) upstream of the firefly luciferase gene (see Methods). The recombinant plasmids contained 1.4 kb of A fragment 5' untranslated sequence, 1.2 kb of B fragment sequence, 3 kb of C fragment sequence and 1.1 kb of D fragment sequence. All of the fragments proved competent to drive transcription of the reporter gene following transfection into HepG2 hepatocarcinoma cells (FIG. 4), suggesting that more than just plasminogen and apo(a) genes might be transcribed from this gene family (see Discussion). Apo(a) gene A fragments were cloned and tested from two individuals. A1 derived from an individual with relatively high plasma Lp(a) concentration (30 mg/ml), while A2 derived from an individual with Lp(a) below the level of detection by ELISA and by Northern blot hybridization. The only DNA differences in the two A fragments are a single base substitution 913 nucleotides before the ATG and nine, rather than eight copies of the repeated sequence TTTTA at position -1373 (see FIG. 3). When normalized to a value of 100% for the D1 fragment, the promoter activity of A1 was 53%, while A2 was only 11%, which is consistent with the lower plasma apo(a) concentration in the A2 individual. In addition, fragment B had 57% relative promoter activity, fragment C had 38%, D2 had 149% and D3 had 23%.
All publications mentioned herein are incorporated by reference to the same extent as though incorporated individually.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2542 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GAATTCATTTGCGGAAAGATTGATACTATGCTTTTATTTTATTTTATTTTATTTTATTTT60ATTTTATTTTATTGAGACTCTCACCCCGGTTGAAGTGCACTGACGTGATTTTGGCTCACT120GCAACTTCCACCTCCTGGGTTCAAGTGAATACTCCAGCCTCCCTAGTAGCTGGGATTACA180GGTGCCCACCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCAC240ATTGGCCTGGCTGGTCTCAAACTCCTGACCTTGTGATCCACCTGTCTTGGCCTCCCAAAG300TGCTGGGATTACAGAGTTGAGCCACCGCACTCGACCCTATGTTTTATTTTTAAAAATATT360TATTTATTTATTTAAGCCACAACTACTAGAATAGGAAGGATTGATATTTTATTAATTTTA420TTTGGTATTTATTATTTTTTTTTCTTTCCTGAGACATTCTTGCTCTGTCACCCAGGCTGG480AGTGCAGTGGCACATTCTTGGCTCACTGCAACCTCCATCTCCTGTGTTCGAGCAATTCTA540GTGCCTCAGCCTACTTAGTAGCTGGGATGACTGGCATGTGCCTCCACACCCAGCTAATTT600TTGTATTTTTTGTAGAGACAGGGTTTTGGCATGTTGCCAGGCTTGTCTCAAACTCCTGGC660CTCAGGTGATCCATCTGCCGTGGCCTCCCAAAATGCTGGGATTATAGGCATGAGCCACCA720CCCCCTCCTGGAAGGATTGATATCTTATAACATAATTTATAATTACAGAAAACATGTGAG780TTCACTAGGAATAAATAAATTTTGAAGATAATAAAAGATTTTCACTTATGTTGTCATTTC840GGCACAGTTTGGTATAGGATGTGGAGATGTTAACATTTATACCTAGCTTGCTCGTAAACT900AAGACCTGAAAGGGTTGTGTCTATCAGCTGCACCCCTGGGTAGCGACACAACCTCGGGAA960GGCCTCAGCCCCCTCCTCGTACAGCACTGCCTGTTGGAAAGCTTGAGGGAGGCTATGGAT1020GTGCAGCACTTGGCAGAGGGTCTGGTCATGGAAGTTACCAGCAAATATGAGCTACTTTTA1080TGATTTTATTTTATCCAAAAGAAAGAGAATGAAAGAAGAGGGGAGGAAACAAGACTAATC1140AGGAAAGATGAAGGTCTAGGGGTGAGGGAAGGAGTAAGGAGACATAAAGGCAATGTGGAG1200CAGCTGAGGGGGGAAATGGCTTTCACCACTTCCCAGCATCTATTGACATTGCACTCTCAA1260ATATTTTATAAGACTCTATATTCAAGGTAATGTTTGAACCCTGCTGAGCCAGTGGCATGG1320GTCTCTGAGAGAATCATTAACTTAATTTGACTATCTGGTTTGTGGGTGCGTTTACTCTCA1380TGTAAGTCAACAACGTCCTGGGATTGGGACACACTTTCTGGGCACTGCTGGCCAGTCCCA1440AAATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTAAGACAT1500AGTTTTTTTAAATTATAAGAATTATTTTTTCTCCCACAATGTAGTAAAAATACATATGCC1560ATGGCTTTATGTGCAATTCATTTAATTTTTGATTCATGAAATTCCCAGTTCAAAATCTTG1620TATATGATTGAAAAATTCTTCCAAAAATTAGTTTAATTTCCCCGTGAAGACTGTCACGGT1680GCTGGAATGAATGGGCAGAAAAAATAATGGTTGATTTTTCTAATCTAAAAGAGTGTGCCT1740ACATGATGGCCAGTCTGGCTGAAAAATAAATAGCCATTGTAGCTAACTATGCAAAGGATG1800GCTAAGCTCTTCGCTTGGTTCTCAGTTTCATTAATTTATATCATCTCTGTTCAGGTGCCA1860TGCTCCCCTCACTAGCAAGTTGAAACAATGAAATAACTCTTTGAATATGTTTGGTTCCTT1920GACCTGTTCATGGAGTGGGACTCAGCATTTCTCTCTTTGTTATGGCCTGAGTAAGGCTTT1980CCATCGGTATACATTTGCTTCTTATCCCTGGAGAAATTATACACATCCATTTGCCAGATG2040ATATACACATATAATGATTCAACAAATACTCAGGGTATTTGTTGAGTGGGTTAGGTCCCC2100ACATTTTTATACATACATACACACATACACACCGTGTGTGATTGTGAATGTAAGTGTGTG2160TCCTTTACAAATACTAGCTTATTTAGCTCATGGTATAGGTAGGGTAGCATAGTCATCCCC2220ATTTTATAAACAAAGAAATCTAGACTTAGGAAAATCATGTTATTTGTCTCGTGACCAAAT2280TCCCAAATCAAGGAAATAAAGAAACCTGGATTTAAGCCAGATTTCCAAGAAAAAATCTAG2340GGCTCTTCTCACTTTTTCATCTTTGTTCCAACATTTGAAAAAATAAATCTAAACACATTC2400CAATGTAACTGAAGAGCAGGTTAATTGTTTGCCACTTGCAGAATCCAATTAAGAAGAGAG2460AAGTCTGGTATAAAGAAAGTGATTTGCTTCCAAAGCTAGCTTAGGGGAAGAAATGCAGCA2520GTCCTGCCGTACCACTTCACTT2542(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GCCTGTTGGAAAGCTTG17(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AGTAGAAGAACCACTTC17(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 15 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:ACCACATGGCTTTGC15__________________________________________________________________________
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Our extended service providers offer an easy to use interactive form online which you may use to request that they initiate a claim on your product. Your other alternative is to call the toll-free number on your extended warranty card or email and have a qualified representative make this request for you with the information you will provide. We believe that our claim initiation request process is the absolute best our industry has to offer. It is designed for simplicity and speed.
Who will perform the service on my product?
Our company works with over 11,000 authorized service professionals. Each technician is extensively trained by the respective manufacturers to provide service on your products. Authorized technicians must complete annual and bi-annual continued education exams in addition to being well versed in manufacturer supplied update bulletins to ensure their level of expertise is as current as the new products which come to market. We urge our customers to provide feedback regarding their experiences which we later use to insure that the level of service we are expected to provide is exactly the level of service you receive.
May I transfer my extended service plan to another person?
Our service plans are 100% transferable. If you would like transfer ownership of the covered product along with its coverage, we require that the request be supplied in writing. For assistance please contact a Representative. Please have all of the new owner's information at hand when requesting a transfer.
Will I have to pay a deductible or any other "hidden costs"?
Generally, the amount of a deductible is almost always proportional to the amount of the premium charged. In order to have a lower deductible, even as low as $0, the plan holder would generally have to agree to a higher premium. For those who want lower premiums, they must agree to a higher deductible. Our extended service plans carry a $0.00 deductible along with low premiums. This means that with an extended service plan there are no high upfront costs and they are committed to cover 100% parts and labor costs on any authorized claim.
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