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Experian Resources
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Clicking. Browsing. Searching. Chatting. Online, mobile, offline. The myraid activities in Asia that are producing potentially valuable data is of an order that is not merely "big", but enormous. Experian commissioned The Economist Corporate Network (ECN) to organise two roundtable lunch discussions with Asia-based c-level executives in Beijng and Singapore. Insights gained from these two events are summarised and commented on in this paper. >> Download Report
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International Holiday Email Performance Study
This study provides global insights to help marketers make adjustments to improve their holiday communication plan and ultimately learn from the past holiday season to make the next year even better. >> Download Report
Email Subject Lines: It's all in the wording
Experian CheetahMail has studied more than 1,000 promotional mailing subject lines and found there are a handful of ways to create crafty versions in order to capture the attention of your specific audience. >> Download Report
The Digitised Pocket: Embracing the Mobile Age
This whitepaper provides insights into the current usage of and attributes towards mobile marketing, the barriers and challengers that marketers face, and their plans to invest in mobile marketing in the coming years. >> Download Singapore Version >> Download Hong Kong Version
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The future of multichannel marketing - Marketer and Consumer Perspectives
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Data Advice for the Modern Marketer
Most marketers see data as a barrier or burden. What they don't recognise is that data can help with the most common problems faced by marketing teams. >> Download Whitepaper
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The Economics of Fraud - Mitigating Risk Amidst Fast Growth and Innovation
Do you know what the fraud hotspots in Asia Pacific are? How are businesses protecting their firms from fraudulent activity and transactions? This 2016 report showed that 59% of firms in Asia Pacific expect fraud to increase in the next 5 years and firms most influenced by fraud tend to be located in the faster growing economies with markets like Indonesia taking the lead. >> Download Report
Analytics: Where are we now
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Improving decisions across the Customer Life Cycle
Companies know that the ability to repeatedly make the right customer decisions, is essential to profitable growth, sharper risk management and higher-performing portfolios. However, volatile, fast-moving markets, economic and regulatory change, and new sources of competition make the right decision a moving target. >> Download Whitepaper
Creating value under Basel III
In this paper, we will provide an introduction to Basel III regulation and discuss some of its impact on banks and the banking system, plus a real business case showing how organizations turn these regulatory challenges into significant business opportunities by optimizing their credit strategies. >> Download Whitepaper
Global Market Update No. 1: The Analytics Issue
In this edition we focus on analytics. The beauty of analytics is the fact that it is real and tangible rather than possible and theoretical, and in this issue we try to show you how to take good advice forward into real results i.e. 'show me the numbers'! >> Download Newsletter
Experian Insight into the changing face of fraud
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Prevention is better than cure
Learn more about the role of a pre-delinquent strategy in managing bad debt. >> Download Report
Report on Debt Management for Telecommunications
Experian delivers a Decision Analytics proposition that addresses the key debt management challenges and enables your business to minimise bad debt, rehabilitate customers rapidly to reduce churn and maintain revenue streams. >> Download Report
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Banks have recognised that managing credit loss and the customer relationship is a challenge and working together with Experian, a solution has been developed to address these key debt management issues. >> Download Report
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Drake and Lil' Wayne at Klipsch
Rappers trade barbs and songs on tour's second night, and one act outshines the other. Chris Shaw photos provided by Klipsch Music Center. David Lindquist / The Star


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Pre-tax profits stood at 17.5 million euros for the period between July and December 2012, up from 16.7 million euros for the same period a year earlier.
Turnover also grew by almost a quarter to 124.1 million euros from 101.4 million in the first half of the 2011/12 fiscal year.
"The figures we announce today are further proof of our continuing and growing financial strength of Borussia Dortmund," said club CEO Hans-Joachim Watzke in a statement.
"But our core principle of legacy before speed will remain."
Dortmund, who almost went bankrupt less than 10 years ago, won back-to-back Bundesliga titles in 2011 and 2012 as well as the German Cup last year.
They are also still in the running in the Champions League and the German Cup though a league title defence this year looks remote with the club in second place, 17 points behind Bayern Munich.
The Ruhr valley club also have the second highest attendance across Europe after Spain's Barcelona, with an average of about 80,000 spectators per home game.
- Sports & Recreation
- Soccer
- Borussia Dortmund


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Release Date:
October 26, 2010
Date Published:
October 26, 2010
Both a practical guide and an inspiring travel gift book, Drives of a Lifetime highlights 400 of the world's best car trips. The eclectic list takes readers across stunning natural landscapes of mountain, coastline, glen, and dale; into charming towns and out-of-the-way hamlets; to palatial estates, lovely gardens, and intriguing historic sites; and through bustling, thriving cities all over the world map. From America's scenic byways to the side lanes of Tuscany and the rugged dirt roads of Australia's outback—and hundreds of inviting routes in between—Drives of a Lifetime shows off the highlights of each area and tells readers exactly how to make the drive themselves. Since roads trips are a beloved cultural touchstone for Americans of every generation, Drives of a Lifetime will capture a huge and diverse audience.
Genre: Transportation, Travel
- Genre: Travel, Transportation
- Publisher: Natl Geographic Society
- Pages: 319
- Language: English
- Release Date: October 26, 2010
other Info.
- Online Item #: 13115018
- Store Item Number (DPCI): 247-05-3285
- Item can be gift wrapped.
- Made in the USA or Imported


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Quédate y te darás cuenta de por qué Negro Bear Inn fue votado como uno de los 10 mejores B & B en California. Gaste su escapada romántica en una pequeña casa de campo de lujo en South Lake Tahoe. La posada cuenta con sólo cinco habitaciones y tres cabañas. Cada habitación tiene un baño privado, cama king size, chimenea, TV / DVD y viene con desayuno completo. Senderismo, ciclismo, y todas las actividades del lago están a poca distancia.
5 en julio 08, 2016
Absolutely my favorite place to stay in South Lake Tahoe. Great, high-end accommodations, outstanding personal, friendly service and 5 star food. Plus a quiet, secluded environment. What more could you ask for?
5 en julio 08, 2016
The atmosphere of Black Bear Inn is terrific. The decor of the lodge and the rooms makes you feel like you are in a high end rustic mountain setting, which you are. The breakfasts each morning were outstanding. Definitely will visit Black Bear Inn again in the future.
4,4 en julio 07, 2016
My husband and I stayed at the inn 4th of July weekend, we were very happy with our stay. The staff upon arrival was so warm and welcoming, the outdoor area is so beautiful I spent some time reading in the garden. The bed was SO comfortable. All and all we will be returning, no doubt about it!
5 en junio 28, 2016
The innkeepers were absolutely wonderful, very detailed, awesome chefs. The lodge was beautiful as were the grounds. I would recommend this B & B and we will be back next year.
4,2 en marzo 31, 2016
We were the only guests at the inn for one night and wanted to stay near the Heavenly California lift so this was perfect. Except that I didn't realize the Frontier Room was on the road: very loud traffic noise most of the evening and it started up again very early in the morning. I wished I had asked to move to a room in the back as the road noise was deafening and we had not brought ear plugs! Otherwise, a good experience: the breakfast was outstanding and the wine and cheese very nice. I would go back but would not choose the Frontier Room again.
4,4 en febrero 25, 2016
My husband and I recently visited the B&B in February to celebrate our anniversary. We have been to the b&b 2 previous times and both times stayed in the main building. This time we took a cabin and although it did not snow (which we had hoped) we wanted to be able to watch the snow. The "Happy Hour" which was wine & cheese. Very enjoyable to sit and chat with the other guests. Breakfast was served every morning. The best day was Sunday as they served Eggs Benedict. The other 2 days were also good. We will be back again hopefully when it snows.
5 en febrero 24, 2016
My husband and I stayed at the Black Bear Inn for a weekend of relaxing and skiing - it couldn't have been more perfect for either. Situated on Ski Run road, the B&B is less than 3 minutes from Heavenly and even has a bus stop outside so you don't need to deal with parking. They have a ski room to store boots, skis and boards in so your equipment doesn't get cold in the car overnight. The B&B itself has been designed with impeccable taste - comfortable and pleasing to the eye, it was hard to get ourselves to leave! We immediately felt at home with Kevin and Jerry - they are phenomenal hosts. The morning breakfasts (8:30-9:30) are to die for, and we loved the fireside wine and cheese (5:00-6:00) chats. We know where we will be staying on our future visits to Tahoe and highly recommend that you make the Black Bear Inn your B&B of choice!
5 en febrero 17, 2016
We were there for Valentines weekend and had a great time. The breakfast was wonderful and the place was immaculate. The 2 gentleman that run the place do an outstanding job.
5 en enero 25, 2016
As a lover of beautiful furnishings, fine food, and stimulating people, we can't say enough good things about Jerry and Kevin's Black Bear Inn. We very much appreciate the combination of their ability to present a AAA experience while making it look so casual and easy. The best recommendation we can give others is to say that we will definately return.
4,4 en enero 13, 2016
Perfect getaway spot. The property is beautifully designed, the service and food exceptional. The owners have good attention to detail and professionalism, with the right amount of customer personalization to make you feel taken care of. If you're looking for a romantic trip in the mountains, this location has great privacy with access to south Lake Tahoe just past the front doorstep.
Reno, NV (RNO-Reno Cannon Intl.) 30 miles
Al circular por la ciudad en Lake Tahoe Blvd, girar hacia las montañas en Ski Run Blvd.. Somos una media milla a la derecha.
Casino, Sala de conciertos, Cenas Elegantes/Gourmet, Lago, Música en vivo, Parques Nacionales, Área natural, Río, Area de esquí
Bed and Breakfast
Jacuzzi/bañera con hidromasaje, Biblioteca, TV por Satélite/Cable, Vídeos, Instalaciones para Bodas
Aire acondicionado, Despertador, Albornoces, Reproductor de DVD, Chimenea, Secador, Servicio de Internet, Tabla de planchar, TV, Wi-Fi
Pistas para bicicletas, Bicicletas, Pasear en barca, Pistas para esquí de fondo, Esquí Alpino, Pescar, Jugar al golf, Pistas para senderismo, Montar a caballo, Pasear en kayak, Pistas para Mountain Bike, Navegar, Comprar, Visitar lugares de interés, Nadar, Deportes acuáticos


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Cancer and occupation in Massachusetts: A death certificate study
Version of Record online: 11 JAN 2007
DOI: 10.1002/ajim.4700060305
Copyright © 1984 Wiley Periodicals, Inc., A Wiley Company
American Journal of Industrial Medicine
Volume 6, Issue 3, pages 207–230, 1984
Additional Information
How to Cite
Dubrow, R. and Wegman, D. H. (1984), Cancer and occupation in Massachusetts: A death certificate study. Am. J. Ind. Med., 6: 207–230. doi: 10.1002/ajim.4700060305
Publication History
- Issue online: 11 JAN 2007
- Version of Record online: 11 JAN 2007
- Manuscript Accepted: 13 APR 1984
Funded by
- NIEHS Occupational and Environmental Health Center. Grant Number: 5 P30 ES 00002
- Abstract
- References
- Cited By
- occupational cancer;
- cancer surveillance;
- occupational cancer surveillance;
- surveillance;
- death certificates;
- mortality odds ratio
This study examines cancer mortality patterns by occupation for white males in Massachusetts using 1971–1973 death records. Its purpose is to identify occupation-cancer associations that, when interpreted in conjunction with results from other studies and hypotheses about potential occupational carcinogens, can serve as leads for more definitive etiological investigations.
Sixty-two malignancy categories (including grouped categories) were investigated for each of 397 occupational categories (including grouped categories) using an age-standardized mortality odds ratio approach.
An important finding was the association between lung cancer and a large number of occupations for which there is support from other epidemiologic studies and/or for which there are reasonable hypotheses as to possible carcinogenic exposures. These occupations include truck drivers, painters, machinists, automobile mechanics, plumbers, cooks, fishermen, heated metal workers, sheet metal workers, and brickmasons/stone-masons/tile setters.


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Malden High senior offensive/defensive lineman Aaron Samano has accepted a scholarship offer from the University of New Haven, Golden Tornadoes head coach Joe Pappagallo told ESPNBoston.com this morning in an email.
The 6-foot-2, 305-pound Samano, who were profiled last summer as part of the high school section's launch, was a two-time Greater Boston League All-Star and captain for the Tornadoes, who went 5-5 this past season. He will be a full-time offensive lineman at New Haven, and will join former Malden teammate Herbans Antenor on the squad.


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Thoroughly enjoyed my stay at The Avalon. The hotel is in the middle of Beverly Hills, but in what felt like a little more of a residential area – which was great because there was very little traffic in the area, especially at night. Lobby is stunning. Very friendly staff, from the valet to check in. I had a room in the tower, and the bed was very comfortable. Loved the mid-century decor of the room. Nice bathroom. I could hear a little noise from hall and room next door, but not enough to bother me or make me lose sleep. Slept very well both nights. Room service was absolutely delicious, and the staff very accommodating when I asked for a very early breakfast. I would definitely stay here again, and have recommended to friends. Love that they even offered yoga by the pool one morning. (The pool is incredible.) If you're considering a stay in Beverly Hills, the Avalon offers a much more personal experience than the big chain hotels in the area.
- Official Description (provided by the hotel):
- Distinguished by its novel physical space, this Beverly Hills California luxury hotel is set around a dramatic pool terrace with private cabanas and the chic Viviane restaurant- all just steps from shopping at the finest boutiques, galleries and restaurants nearby.Avalon Hotel Beverly Hills offers luxurious amenities, stylish accommodations, and creative meeting and event space in the heart of Beverly Hills, California.Inspired by the relaxed "patio lifestyle" of mid-20th Century Southern California, Avalon's 84 Beverly Hills hotel rooms, suites and penthouse studios offer guests comfort and style in the heart of Beverly Hills.Custom-designed and classic furnishings by Kelly Wearstler. Pampering Beverly Hills accommodations include custom furnishings and beds with sumptuous Italian designer lines and Neil George amenities. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Hotels.com, Priceline, TripOnline SA, Expedia, Hotwire, Orbitz, Odigeo, Travelocity, HotelTravel.com, Cheap Tickets and Jetsetter so you can book your Avalon Hotel Beverly Hills reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Avalon Beverly Hills
- Beverly Hills Avalon Hotel


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|Release Year
|From the cardback:
The bounty hunter Jango Fett is a shrewd mercenary mysteriously linked to the growing Separatist army and the unexplained assassination attempts on Senator Amidala. Armed with dual pistols and a jet backpack, he is always prepared for dangerous confrontations. Jango is equally as dangerous in space as he pilots his modified starship, Slave I, to capture or destroy anything that gets in the way.
|Special Thanks
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Ratings based on 1,079 Verified Reviews
They paid and stayed. We double-checked.
4.4 Room cleanliness
4.8 Service & staff
4.4 Room comfort
4.0 Hotel condition
Expedia Verified ReviewsThey paid and stayed. We double-checked.
by A verified traveler
super hotelPosted Apr 29, 2016
Pros: emplacement
Location: Hollywood boulevard, proche d'universal studios
tres bon emplacement. grande suite avec chambre, salon et cuisine équipée; bon petit déjeuner continental compris. parking couvert à 13 dollars la nuit.
by Jose from Asheville, NC
Great for families, highly recommendedPosted Apr 1, 2016 on Hotels
We absolutely love the Magic Castle hotel. I have to admit, we were nervous at first because it felt dated. I believe it is an older converted motel. Regardless, once we interacted with the friendly staff and took advantage of all their offerings, we were sold. If we ever stay in Hollywood, it will definitely be at the Magic Castle Hotel. Why? The rooms are spacious, the staff is incredibly helpful and friendly. They treat our kids like the king and queen of the place. Snacks are made available to them at no charge and at any time. We had a fantastic dinner up the street at Yamashiro and the staff shuttled us there and picked us up. The few nights we stayed, we woke up to breakfast right outside in their patio by the pool. A magician greeted our kids and kept us entertained. Once we wanted to hit the town, we were just a few blocks away form the hollywood walk of fame, a short walk to the hollywood bowl, and a few minutes from many restaurants. We love it.
by A verified traveler
Exceptional service.Posted Mar 27, 2016
The hotel has a cool 50's feel and the service is like what you would expect from back then: the staff is incredibly friendly and helpful and go out if their way to make your stay as comfortable as possible. I would absolutely recommend this hotel. Also, take advantage of the ability to have an "invite" to the Magic Castle itself - it's an amazing evening.
by A verified traveler
Posted Mar 16, 2016
Excellent staff. Room needs updating. Great location but is noisy
by A verified traveler from San Francisco
Fine Hotel in a good location.Posted Mar 16, 2016
Pros: Lots of free snacks.
Cons: Don't charge for water when you are giving so much away.
Location: A block from Hollywood Blvd and the Hollywood Walk of Fame as well as the Dolby Theater where the Academy Awards are held.
Staff was extremely accommodating. Hotel was clean. 24 hour swimming pool was nice. Sorry I did not take advantage of the Magic Castle Club next door.
by wang from 中国
服务不错Posted Mar 16, 2016 on Hotels
服务非常热情,有免费早餐 住了两次,第一次住的标准单间,房间不是很大,床垫有点太高了,另外倒时差的原因,没有睡太好 第二次住的是套间,房价没有高太多,但是多了阳台,客厅,厨房,假如想舒适一点的话,建议大家订套间,性价比比单间高很多
by wang from 中国
服务非常好Posted Mar 16, 2016 on Hotels
by Michael
Posted Mar 15, 2016 on Hotels
by A verified traveler
Posted Mar 14, 2016 on Hotels
by Mathias
Posted Mar 13, 2016 on Hotels


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Butterfly: Marsh Commodore
Find, identify & discuss the insects of SANParks
First unread post • 1 post • Page 1 of 1
- Johan van Rensburg
- Legendary Virtual Ranger
- Award: Birder of the Year (2015)
Award: Birder of the Year (2014)
- Posts: 2316
- Joined: Wed Sep 06, 2006 3:33 pm
- Location: Jam Street
Butterfly: Marsh Commodore
Large view
The Marsh Commodore, Precis ceryne ceryne, is found from December to June, from two distinct broods that overlaps during autumn. Usually seen on flowers and food plants near water. Sits on grass stems from where it chases other butterflies. Seen near Witbank.
679 Last 5 lifers: Rüppell's vulture, Spotted crake, Lesser jacana, Burchell's courser, Double-banded courser
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Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
► Nascent RNA forms hybrids with underwound DNA upstream of elongating Pol II ► Single-stranded DNA so formed is prone to damage which results in genome instability ► Sen1 helicase acts to remove R loops by resolving RNA:DNA hybrids ► Sen1 function in Pol II elongation and termination may relate to R loop resolution
In S. cerevisiae nascent transcripts formed by RNA polymerase II (Pol II) on protein-coding genes are immediately processed, packaged, and exported to the cytoplasm (Luna et al., 2008; Moore and Proudfoot, 2009). Messenger RNA (mRNA) packaging protects transcripts from degradation, but also the DNA template from invasion of nascent RNA into the DNA duplex behind elongating Pol II (Aguilera and Gómez-González, 2008). The resulting RNA:DNA hybrid exposes single stranded (ss) nontemplate DNA, a structure referred to as an R loop. R loop formation has been associated with increased occurrence of transcription-associated mutation (TAM) or recombination (TAR), presumably because both induced and spontaneous lesions are more likely to occur on ssDNA. Thus, deletion of genes encoding the THO (Thp2, Hpr1, Mft1, and Tho2) and THSC or TREX-2 (Thp1, Sac3, Sus1, and Cdc31) complexes required for mRNP formation in S. cerevisiae—or, similarly, the splicing factor ASF/SF2 in metazoans—increase levels of R loop formation and consequently TAM and TAR (Chávez et al., 2000; Fischer et al., 2002; Gallardo and Aguilera, 2001; González-Aguilera et al., 2008; Huertas and Aguilera, 2003; Li and Manley, 2005). R loop formation in these mutants may also be connected to Pol II stalling, consequently interfering with processive elongation (Mason and Struhl, 2005; Rondón et al., 2003) and RNA processing (Libri et al., 2002; Rougemaille et al., 2008). Similarly, DNA replication may be compromised when replication forks encounter R loops or a stalled Pol II (Wellinger et al., 2006).
Although little is known about R loop resolution in yeast, in mammals their formation and resolution play a productive role in the stimulation of class switch recombination (CSR) and somatic hypermutation (SHM) in clonally expanding B cells (Yu et al., 2003). Both processes are initiated by activation induced deaminase (AID) (Muramatsu et al., 1999). Double-strand breaks (DSBs) subsequently trigger CSR via nonhomologous end joining (NHEJ) (Yu and Lieber, 2003). Although S. cerevisiae does not express AID, ectopically expressed AID can recognize ssDNA in R loops as a substrate when expressed in mRNA packaging mutants (Gómez-González and Aguilera, 2007; González-Aguilera et al., 2008).
Many events during transcription are orchestrated by proteins binding to the carboxy-terminal domain (CTD) of the Pol II largest subunit. CTD consists in yeast of 26 hepta-peptide repeats (YSPTSPS) that are dynamically modified during transcription. In particular, serine phosphorylation occurs during early (ser5-, 7-P) and late (ser2-P) elongation phases to allow stage specific binding of elongation and RNA processing factors (Kim et al., 2009; Komarnitsky et al., 2000). Transcription termination is also directed by different CTD-bound proteins that recognize specific sequences on the emerging nascent RNA. For protein-coding genes, this requires polyA (pA) site recognition by a ser2-P CTD bound multicomponent cleavage and polyadenylation complex (CF IA/B and CPF), as well as degradation of the downstream RNA by Rat1 exonuclease (Gross and Moore, 2001; Kim et al., 2004b; Meinhart and Cramer, 2004).
Termination of many noncoding RNAs requires an additional component, the NRD complex (Sen1, Nab3, and Nrd1), in which Nrd1 is bound to ser5-P CTD (Steinmetz et al., 2001; Vasiljeva et al., 2008). NRD-dependent termination also requires recognition of frequent short RNA sequences by Nrd1 and Nab3 (GUAA/G and UCUU respectively) (Carroll et al., 2007), although the exact sequence and NRD component requirements may vary for different terminators (Kuehner and Brow, 2008). Nrd1 ser5-P CTD specificity confines this termination pathway to transcriptional stages in which ser5-P CTD prevails and leaves promoter distal Nrd1/Nab3 binding sites unrecognized (Arigo et al., 2006a; Gudipati et al., 2008). Furthermore, as Nrd1 interacts with the exosome, NRD-terminated RNA is either degraded to protein protected stable transcripts (e.g., snoRNAs) or completely, as is the case with cryptic unstable transcripts (CUTs) (Arigo et al., 2006b; Thiebaut et al., 2006; Vasiljeva and Buratowski, 2006). Recent genome-wide transcription profiling studies reveal the wide extent of CUTs produced by Pol II and terminated by NRD. This further emphasizes the biological importance of NRD-dependent termination (Neil et al., 2009; Xu et al., 2009). Importantly, both termination pathways can substitute for each other and so provide mutual fail-safe termination mechanisms (Kim et al., 2006; Rasmussen and Culbertson, 1998). Thus, NRD termination is also important to rescue polymerases that fail to terminate at a polyA signal, especially on highly transcribed genes. Interestingly, these genes show a particular requirement for Sen1 (Rondón et al., 2009).
SEN1 codes for a 240 kDa superfamily I helicase (DeMarini et al., 1992), and its S. pombe homolog possesses 3′-5′ nucleic acid unwinding activity (Kim et al., 1999). The essential C terminus contains the helicase domain, a nuclear localization sequence (NLS), and a domain necessary for interaction with the Glc7 phosphatase component of CPF (Nedea et al., 2008; Ursic et al., 1995; Winey and Culbertson, 1988). The Sen1 975 N-terminal amino acids, although dispensable for growth, interact with Pol II, RNase III endonuclease Rnt1, and the nucleotide excision repair endonuclease Rad2 (Ursic et al., 2004). Mutation of the Sen1 helicase domain results in direct and indirect pleiotropic defects in transcript processing and termination, leading to a perturbed genome-wide profile of Pol II and defective Pol I transcription termination (Kawauchi et al., 2008; Rasmussen and Culbertson, 1998; Steinmetz et al., 2001, 2006; Ursic et al., 1997). Although the severe character of these phenotypes may be explicable by the limiting presence of Sen1 in NRD (as it is only present at 125 copies/cell) (Ghaemmaghami et al., 2003), they have not been clearly attributed to a molecular function of Sen1. Employing the temperature sensitive sen1-1 mutant (helicase domain G1747D), we set out to characterize the molecular role of Sen1 in transcription termination. We now identify broad functions for Sen1 during Pol II transcription in reducing R loop formation and consequent prevention of transcription-associated genome instability.
Mutation of the Sen1 helicase domain results in genome-wide transcription termination defects of noncoding RNAs, but also of some protein coding genes (Steinmetz et al., 2006). Thus, when tested by transcription run on (TRO) experiments with the plasmid gene construct KGG (Figure 1A), with the KanMX4 gene terminated by the weak GAL10 pA signal (Morillon et al., 2003), sen1-1 mutants grown for 150 min at nonpermissive temperature (37°C) show a strong termination defect (Figure 1A, upper panels) (Rondón et al., 2009). This indicates either a requirement for Sen1 in Rat-dependent termination or that some transcripts over the weak GAL10 pA signal are terminated by the NRD failsafe termination mechanism.
To determine whether Sen1 protein-protein interactions or its helicase function are required for transcription termination, we repeated TRO analysis in WT and sen1-1 cells transformed with additional Sen1 expression constructs. Transcribed from an ACT1 promoter, these either contained the NLS and the Glc7 interaction domain [Sen1(323)] or additionally the C-terminal helicase domain [Sen1(1212)] (Nedea et al., 2008). As shown in Figure 1A, Sen1(1212) but not Sen1(323) rescued the sen1-1 termination defect, implying that the sen1-1 termination defect is caused by loss of helicase function and not Glc7 mediated recruitment of CPF. We also examined steady-state mRNA produced from the KGG construct (Figure 1B). mRNA levels were reduced in sen1-1 cells and partially complemented by coexpression of Sen1(1212), but not Sen1(323). Similarly, coexpression of Sen1(1212) restored wild-type levels of endogenous PMA1 mRNA, also previously shown to display mild termination defects in sen1-1 (Kawauchi et al., 2008).
The above results indicate that the Sen1 helicase domain is required both for efficient Pol II termination and mRNA accumulation. As these effects could be attributed to defective 3′ end processing, we employed an in vitro cleavage and polyadenylation assay using CYC1 3′ flanking RNA as the pA substrate (Figure 1C). sen1-1 shows no defects in RNA 3′ end processing. Confirmation of this result is provided by reverse transcription analysis of ACT1 pA usage, in which sen1-1 showed WT pA selection (Figure S1 available online). In contrast, a CF IA mutant strain, rna14-1, showed the expected defect in both in vitro 3′ end processing and in vivo pA selection (Figure 1C and Figure S1). Finally, like sen1-1, the rat1-1 termination mutant (or both combined) had no effect on mRNA 3′ end formation but stabilized the 3′ end cleavage product, indicative of loss of exonuclease "torpedo" function (Kim et al., 2004b; Luo et al., 2006). Overall, these combined analyses show that the Sen1 helicase is dispensable for 3′ transcript processing but is required to promote transcriptional termination.
Since S. pombe Sen1 can use RNA:DNA hybrids as an in vitro substrate (Kim et al., 1999), we considered the possibility that Sen1 may remove RNA:DNA hybrids formed by nascent RNA and the template strand. Such hybrids were previously shown to form in THO mutants, causing increased rates of transcription associated mitotic recombination (Huertas and Aguilera, 2003). RNA:DNA hybrids may also be naturally encountered in transcribed regions downstream of pA signals, where THO is undetectable on chromatin (Kim et al., 2004a; Luna et al., 2005). We therefore tested whether sequences downstream of a pA signal elicit TAR in sen1-1. We employed a plasmid borne recombination substrate that carries two truncated regions of LEU2 overlapping by 600 nt of homologous sequence (LNA). Lack of THO elicits TAR in LNA and consequent restoration of LEU2, as previously shown (Figure 2A) (Prado et al., 1997). In contrast, when transcription between both repeats is terminated by insertion of the CYC1 38nt pA signal (CYC1t, LNAT), recombination levels in the hpr1Δ strain were reduced to background WT levels, presumably because Pol II termination restricts R loop formation. Similar analysis of LNA and LNAT transformed rat1-1 and rna14-1 showed no detectible increase in recombination, confirming that defects in CPF/Rat1 dependent transcription termination per se do not promote recombination (Luna et al., 2005). In marked contrast, sen1-1 transformed with either LNA or LNAT showed high levels of recombination, suggesting that RNA:DNA hybrids may form throughout the mRNA coding region irrespective of the CYC1t. This lack of CYC1t suppression reiterates the sen1-1 CYC1 termination defect previously reported (Kawauchi et al., 2008; Steinmetz et al., 2006). Moreover, the fact that CYC1t (in LNAT) further stimulated recombination may reflect an increase in R loop formation downstream of pA signals.
To determine whether this recombination phenotype was specific to Sen1, we similarly tested other NRD complex components. Although recombination levels were somewhat increased in nab3 and nrd1 CTD-interacting domain mutants (but not the RNA binding domain mutant nrd1-102) transformed with LNA, they were reduced to background levels in LNAT (Figure 2A). This suggests that these NRD mutants still recognize the CYC1t. Why these NRD mutations elicit some recombination is unclear at this point, but may reflect alteration in mRNP biogenesis. The fact that CYC1t abrogates recombination in NRD mutants but stimulates recombination in sen1-1 clearly separates Sen1 function from Nab3 and Nrd1 and argues that Sen1 plays a distinct role outside the NRD complex.
Hyperrecombination in THO and THSC/TREX-2 mutants shows clear transcription dependence, as it increases with greater transcript length and transcription rate but decreases when the R loop-forming RNA is removed either by RNase H activity or ribozyme directed RNA cleavage (González-Aguilera et al., 2008; Huertas and Aguilera, 2003). To test whether sen1-1 shows a similar transcription-dependent recombination phenotype, we analyzed sen1-1 recombination levels for various direct-repeat recombination substrates. As shown in Figures 2B and 2C, levels of recombination in sen1-1 correlate with the length and transcriptional rate of the gene. Thus, TAR, although not abolished, was significantly decreased in two different recombination substrates when transcription from a GAL1 promoter was glucose repressed. Finally, we verified that recombination was also stimulated between direct repeats in a chromosomal context (Figure 2D). These observations suggest that there is a correlation between transcriptional activity and sen1-1 recombination levels.
Evidence for RNA:DNA hybrid accumulation in THO mutants derives from expression of the human AID in yeast which was shown to cause a 25-fold increase in TAR (Gómez-González and Aguilera, 2007). Employing 5′ and 3′ truncated overlapping GFP repeats and intervening LacZ sequence as a recombination substrate, FACS analysis of GFP-positive cells showed that AID expressed in sen1-1 also stimulates recombination albeit only 2.5-fold (P[Wilcoxon-rank-sum test] = 0.017; Figure 3A). As discussed below, this moderate but significant stimulation of TAR by AID could reflect the nature of RNA:DNA hybrids formed in sen1-1 or be due to the fact that recombination levels in sen1-1 cells that did not express AID were already very high.
AID C to U deamination preferentially occurs in a WRC (or GYW on the opposite strand) sequence (Pham et al., 2003). In regions of R loop formation, AID has access to the nontranscribed strand (NTS), although some mutations occur on the transcribed strand (TS) (Gómez-González and Aguilera, 2007). In an attempt to analyze the nature of AID-induced mutations in sen1-1, we transformed WT and sen1-1 cells (both ura3–) with plasmid-encoded LACZ::URA3 chimeric gene (pLAUR, Figure 3B) and selected AID-induced ura3 mutations on 5-FOA (5-fluoorotic acid). Although mutation rates were very low in WT and sen1-1 cells grown at semipermissive temperature, AID expression in sen1-1 significantly increased mutation rates (Figure 3B). DNA sequencing of 57 5-FOA-resistant transformants revealed that 42% carried a point mutation in URA3, of which 71% had a point mutation within an AID sequence motif. Of these mutations 70% occurred on C (12; Fisher's test; p < 0.003) or the NTS and only 30% (five; p < 0.05) occur on G or the TS (Figure 3C and Figure S3). The small amount of WT transformants sequenced showed a distribution as earlier reported (Gómez-González and Aguilera, 2007). Since AID expression not only increased the amount of point mutations within the transcribed URA3 in sen1-1 but also preferentially acted on the NTS, this suggests that R loops are formed during transcription in sen1-1 cells and displace the nontranscribed DNA strand.
We next tested whether the high recombination levels seen in sen1-1 were sensitive to RNase H overexpression as previously observed with THO and THSC/TREX2 mutants and indicative of increased R loop formation (González-Aguilera et al., 2008; Huertas and Aguilera, 2003). Thus, RNase H overexpression (from pRNH201) reduced recombination rates of pLLacZ in sen1-1 by 5.6-fold (Figure 3D). Furthermore, the sen1-1 recombination phenotype correlated with deficient helicase activity and could be rescued by high copy expression of pYsen1, encoding for the Sen1 helicase domain. To exclude the possibility that increased recombination levels in sen1-1 could be a consequence of the sen1-1 transcription termination defect, rather than the ability of Sen1 to remove R loops, we tested the capability of Sen1 helicase domain to suppress the hyperrecombination phenotype of the THO mutant mft1Δ, which displays no transcription termination defect but increased levels of R loops. As shown in Figure 3E, overexpression of the Sen1 helicase domain in AID- and pGLG-transformed mft1Δ cells caused a substantial reduction in the number of GFP-recombinant cells. We conclude that Sen1 enzymatic activity is able to directly restrict cotranscriptionally formed R loops.
To obtain independent evidence for the existence of R loops formed in sen1-1, we employed both DNA (DIP; Figure 4A) and chromatin immunoprecipitation analysis (ChIP; Figure S4A) with the RNA:DNA hybrid-specific antibody (S9.6) (Hu et al., 2006). Yeast transformed with pGLLacZ displayed hybrid signal over the recombining LEU2 sequence in sen1-1 but not WT cells when shifted for 1 hr to nonpermissive temperature and induced by galactose. Hybrid signal then diminished to WT background levels when transcription was repressed by glucose addition to the medium (Figure 4A). Notably, the untranscribed origin region of pGLLacZ gave background signals (Figure S4B). Furthermore, where DIP signals were detected on LGZ2 and LGZ5 in sen1-1 grown in galactose, these signals were sensitive to RNase H digestion prior to immunoprecipitation (Figure S4C). Overall, these data demonstrate that R loop formation is highly dynamic, closely following transcriptional activation and repression of pGLLacZ. Parallel experiments with the THO mutant strain hpr1Δ gave significant but lower levels of hybrid signal over the 5′ positioned LEU sequence (LGZ2) but not over LGZ5 (Figure 4A). The 3′ LGZ5 probe spans the LEU2 pA signal and so will lack transcripts in hpr1Δ due to Pol II termination. sen1-1 in contrast is termination defective so that read-through transcripts in this strain will still elicit R loop formation.
The S9.6 antibody was also employed in a regular ChIP analysis of endogenous PMA1 and compared to Pol II ChIP profiles. RNA:DNA hybrid signal was detected over PMA1 (Figure S4A), although general signal intensities were lower than those obtained by DIP analysis of the highly expressed pGLLacZ. When normalized to the gene 5′ end in WT and sen1-1 cells, signals were detected wherever Pol II was present on chromatin (Kawauchi et al., 2008). Of note, in chromatin isolated from rna14-1 that shows no recombination phenotype, little hybrid could be detected downstream of the pA signals, even through Pol II is still present at these positions. This may indicate that while hybrid is degraded downstream of the pA signal in presence of Sen1 and Rat1, it is stabilized in rat1-1 sen1-1 and to a lesser extent in sen1-1. A modest accumulation of hybrid signal was observed over the pA signal compared to the gene body (P6 versus P5) in all tested strains and may indicate a region that is particularly prone to form RNA:DNA hybrids.
In view of the only modest RNA:DNA hybrid accumulation observed over PMA1, we developed an independent assay for R loop formation over this same gene (Figure 4B). Genomic DNA with associated nascent transcripts was isolated from WT and sen1-1 cells, shifted for 1 hr to 37°C, and subjected to RNase H digestion. Subsequently, DNA was digested with DNase I and nascent RNA that survived this treatment was detected by RT-PCR with RT primers either within PMA1 (P5) or downstream of the pA signal (P7). As shown in gel fractionation of these amplified DNAs, sen1-1 chromatin associated RNA is highly sensitive to RNase H treatment. Quantitative PCR (qPCR) values (ratio of RNase H sensitive to total signal) show that although in sen1-1 chromatin associated RNA is nearly fully RNase H sensitive, in WT RNase H sensitivity increases downstream of the PMA1 pA signal. This suggests that in sen1-1 a higher fraction of RNA forms R loop structures with genomic DNA, whereas in the WT R loops are particularly prone to forming downstream of the pA site.
From these combined analyses, we conclude that R loops accumulate in sen1-1 in a transcription-dependent manner. Furthermore, they imply that WT-transcribed and subsequently packaged RNA still forms some level of RNA:DNA hybrids. The amount of RNA in R loop conformation appears to differ throughout the transcribed PMA1 gene, with a particular prevalence over the pA site.
Experiments presented so far suggest that R loops formed in sen1-1 elicit TAR. However, to substantiate this conclusion and further exclude the possibility that sen1-1 TAR is an indirect consequence of its transcription termination defect, we employed genomic analysis. A comparison of RNA steady-state levels of sen1-1 to WT cells grown for 150 min at 30°C was performed by hybridization to strand-specific tiling arrays (data available at http://www.ebi.ac.uk/arrayexpress/ with accession number E-TABM-863) (David et al., 2006). This revealed that among the transcripts whose expression was significantly changed (adjusted p value of < 0.01), stable unannotated transcripts were overrepresented (19% stable unannotated transcripts versus 7% for open reading frames [ORFs]), confirming a Sen1 role in the termination of some of these genes (Figure S5A and Table S4). Importantly, THO or THSC genes were absent among the 347 ORFs that were significantly changed, excluding the possibility that R loops are formed in sen1-1 as an indirect consequence of alteration in THO or THSC/TREX-2 expression. We next considered whether the 347 significantly changed ORF transcript levels correspond to DNA damage repair and cell-cycle (DDCC) genes. DDCC genes were underrepresented, and among those changed, no clear trend was observed (Figure S5B and Table S4), with some mildly upregulated (i.e., REC104, POL4, SMC5, SCM4, and TOP2) and others downregulated (i.e., DIA2, MMS2, and NEJ1).
To define which DNA repair mechanism was induced by R loops formed in sen1-1, we searched for synthetic genetic interactions of sen1-1 with mutants of candidate genes involved in either NHEJ or homologous recombination (HR). At permissive temperature (25°C) or in presence of replicative stress, we observed genetic interaction with various factors involved in HR but not in NHEJ (Figure 5). Thus, sen1-1 (but not nrd1-102; Figure S5C) causes synthetic lethality with rad50 and mre11 deletion mutants, both found in a complex with Xrs2 and involved in initial recognition and ss resection at a DSB (Figure 5A). The critical need for HR in sen1-1 cells is demonstrated by the phenotypes of double mutants of sen1-1 and rad52Δ, sgs1Δ, srs2Δ, or mus81Δ. These all showed synthetic defects or displayed increased sensitivity to growth in hydroxyurea (HU). In contrast, sen1-1 yku70Δ double mutants showed neither growth defects nor increased sensitivity to HU, suggesting that NHEJ is not required for cell survival of sen1-1 cells (Figure 5B). Taken together, these data suggest that proteins involved in HR but not NHEJ are important to maintain sen1-1 viability.
In cells that accumulate DNA damage, factors involved in DNA repair are rapidly recruited to the damage site (Lisby et al., 2001). Consistent with the role of R loops in DNA damage as seen in sen1-1, we observed that many sen1-1 cells display an accumulation of GFP-tagged Rad52 (encoded on pWJ144; Figure 6A). This percentage of sen1-1 cells forming foci increased from 8% at 25°C to 13% when cultures were shifted to semipermissive (30°C) or nonpermissive (37°C) temperature for 3 hr. In contrast, only about 1% of WT cells formed foci under these conditions. Importantly, steady-state RNA analysis from the same cell populations showed that Rad52 foci formation correlated with accumulation of a bicistronic transcript from the SNR13 snoRNA gene, which results from termination at the next available pA when NRD-dependent termination is defective (Rasmussen and Culbertson, 1998). As shown in Figure 6A (right panels), the SNR13-TRS31 transcript is already present in sen1-1 at 25°C but increases over time at 30°C and to a greater extent at 37°C. These results connect the various sen1-1 phenotypes observed in this work and support the assumption that they are caused by mutation of Sen1 helicase domain in sen1-1.
Using this assay as an indicator of ongoing HR in sen1-1, we reinvestigated the role of Sen1 helicase in preventing or resolving transcription dependent R loops. WT and sen1-1 transformed with Rad52-GFP and pYSen1, which encodes the Sen1 helicase domain transcribed from the GAL1 promoter, were grown in raffinose as a neutral carbon source for 1 hr at 30°C. After addition of glucose or galactose to respectively repress or activate Sen1 helicase expression, the amount of Rad52 foci forming cells was counted. Expression of the Sen1 helicase domain rapidly reduced the number of sen1-1 nuclei displaying Rad52 foci to almost WT levels (Figure 6B). Employing a sen1-1 rpb1-1 double mutant, which allows rapid transcription shutdown at the nonpermissive temperature due to a mutation in the Pol II largest subunit (rpb1-1) (Ursic et al., 2004) (Figure 6C), we could also correlate the formation of Rad52 foci to transcriptional activity. When grown at 37°C, Rad52-GFP transformed sen1-1 rpb1-1 cells displayed a time-dependent decrease in Rad52 foci as compared to growth at 25°C. Since sen1-1 cells accumulate Rad52 foci when shifted to 37°C, these data indicate that upon transcription shutdown, Rad52 foci disappear either as DNA damage is immediately repaired or the cells die.
The deleterious effects of transcription on genome integrity have been suggested by various observations (Aguilera, 2002). Whenever the integrated process of transcript processing, packaging, and export in eukaryotes is disrupted, genome instability can be observed (Baaklini et al., 2004; Broccoli et al., 2004; Jimeno et al., 2002; Luna et al., 2005). This has been shown to derive from R loops, which preferentially form when mRNP biogenesis is disrupted. Our data show that Sen1 helicase plays a pivotal role in the prevention of genome instability by recombination. A large fraction of this instability is transcription dependent and linked to the formation of R loop structures. The exact nature of these structures remains to be established, but we show their accumulation can still occur with normal mRNP biogenesis. If these structures are not removed by either Sen1 helicase or RNase H directed degradation, they can exert a deleterious effect on genome stability, as is further illustrated by SEN1 genetic interaction with HR genes. The occurrence of Rad52 foci, as a marker for ongoing recombination, shows that recombination is related to transcription, as well as to the presence of a functional Sen1 helicase domain. In summary, we suggest that R loop formation is more frequent than hitherto anticipated and requires active removal by helicases such as Sen1.
We suggest that as soon as the nascent transcript emerges from the polymerase body, mRNA packaging and R loop formation occur in kinetic competition (Figure 7). A fragile equilibrium between protective mRNA packaging and the hiding of specific recognition sequences is likely to exist (Bucheli and Buratowski, 2005; Bucheli et al., 2007). Therefore, RNA packaging is likely to be incomplete so leaving some transcript available for R loop formation. For pervasive CUT transcription, termination depends on NRD and by inference on Sen1 (Arigo et al., 2006b). If R loops formed in sen1-1 extend to many CUT loci, then their accumulation, even if transient, would cover substantial regions of the genome. In both CUTs and mRNA coding genes, R loops could interfere with DNA replication, induce ssDNA breaks, or be recognized as recombination intermediates. Any of these possibilities could explain the essential need for DSB sensing proteins in sen1-1 (Figure 5). However, the different genetic interactions of sen1-1 and hpr1 with HR or S phase checkpoint genes suggest structural and functional differences of the replication/recombinogenic intermediates that are formed (Gómez-González et al., 2009). Alternatively, these differences may hint at a transcription-independent role of Sen1 in DNA damage repair that is yet to be uncovered.
How may R loop accumulation in sen1-1 be related to its transcription termination defect? R loops were originally hypothesized to slow down transcription elongation, thereby enhancing termination (Proudfoot, 1989). This would give time for the Rat1 5′-3′ exonuclease "torpedo" to catch up with Pol II but would require R loop resolution by an enzymatic activity such as Sen1 prior to degradation. Based on observations made on THO mutants, R loops have been suggested to interfere with transcription elongation (Huertas and Aguilera, 2003; Mason and Struhl, 2005). Employing sen1-1, in which transcript processing is normal, we predict that reduced steady-state RNA accumulation is due to reduced transcript elongation. Furthermore, the data presented here support the view that R loops preferentially form in termination regions. Thus, we employed the LNA/LNAT recombination substrates, anticipating that even though the CYC1 pA would not elicit termination (Kawauchi et al., 2008), it should serve as a 3′ processing signal (Figure 1C), promoting disassembly of THO and consequent R loop formation (Kim et al., 2004a). Compared to LNA, sen1-1 recombination levels increased 2-fold in LNAT. This demonstrates for sen1-1 in contrast to THO mutants, that RNA cleavage in the context of a pA is not sufficient to relieve recombination. To reiterate this point, Figure S2 shows that in a ribozyme containing substrate, recombination levels in sen1-1 are reduced similar to hpr1Δ (Huertas and Aguilera, 2003). As both ribozyme cleaved ends are unprotected they are likely to be degraded and so reduce R loop forming substrate. However, this appears not to be the case if RNA in sen1-1 cells is cleaved at a pA, possibly as RNA downstream to the polyA signal may be less packaged and so temporarily protected from degradation by R loop formation. Although these studies require a more detailed biochemical analysis, we predict from these initial results that R loops may play a role in transcriptional termination.
In summary the molecular and genetic effects of Sen1 inactivation presented here reveal that Sen1 acts to protect the heavily transcribed genome from R loop-mediated DNA damage. Of note, mutations in the helicase domain of the human SEN1 gene ortholog SETX (encoding Senataxin) cause the neurodegenerative diseases, Ataxia with Oculomotor Apraxia Type II (AOAII), and juvenile amyotrophic lateral sclerosis (ALS4). Like sen1-1 these SETX mutants show defects in transcription, RNA processing, and DNA damage repair (Moreira et al., 2004; Suraweera et al., 2007, 2009). It remains to be established whether the tendency of transcription to induce R loop formation is a general feature of all eukaryotic genomes. It is possible that a range of dedicated helicases act to resolve these potentially harmful structures.
Yeast strains, plasmids, and primers are listed in Tables S1–S3. Standard methods are detailed in the Supplemental Experimental Procedures. In outline, genetic crossing of single mutants (sen1-1 and nrd1-102) with HR and NHEJ mutants employed standard conditions. Recombination frequencies were scored by counting of LEU+ cells or by FACS analysis of GFP+ cells. GFP-Rad52 nuclear foci were detected by epifluorescent microscopy.
The transcription run on (TRO) method and probes for pKGG are as described (Morillon et al., 2003; Rondón et al., 2009).
For RNA isolation, strains were grown in minimal (selective) media at indicated temperatures. After acidic phenol RNA isolation, RNA (15 μg) was separated by 1% formaldehyde agarose gel electrophoresis. RNA immobilized on nitrocellulose membranes was detected with random primed probes.
ChIP employed real-time qPCR as previously described (Rondón et al., 2009). S9.6 purified antibody was employed for immunoprecipitation and was a kind gift from Stephen Leppla (Hu et al., 2006) and Antonin Morillon (Institut Curie, Paris). For DIP analysis, sonicated, deproteinized chromatin was immunoprecipitated with S9.6 antibody and amplified by qPCR as further detailed in the Supplemental Experimental Procedures (Liu et al., 2005). RNase H sensitivity was measured by treatment with RNase H prior to immunoprecipitation.
Sequential RNase H (NEB, 2 hr at 37°C) and DNase I (Roche, 4 hr at 37°C) digestion of genomic DNA (5 μg) isolated from logarithmic phase cells cultivated for 1 hr at 37°C with yeast breaking buffer (2% [v/v] Triton X-100, 1% [w/v] SDS, 100 mM NaCl, 10 mM Tris [pH 8.0], and 1 mM EDTA [pH 8.0]), phenol, and glass beads. Isolated RNA was reverse transcribed (Invitrogen Superscript III) according to the manufacturer.
These are presented in the Supplemental Experimental Procedures and include in vitro 3′ end processing, 3′end RACE, and microarray analysis.
Thanks to our lab colleagues, especially Monika Gullerova and Jurgi Camblong, Julien Gagneur, Sandra Clauder-Muenster, and Charles Giradot (submission to ArrayExpress). Funding was from the Wellcome Trust to N.J.P., the Spanish Ministry of Science and Innovation (BFU2006-05260 and Consolider Ingenio 2010 CSD2007-0015) and Junta de Andalucía (BIO102 and CV2549) to A.A., and a joint ESF grant to N.J.P. and A.A. (NuRNASu). H.E.M. was recipient of a B.I.F. studentship and a European Molecular Biology Organization (EMBO) short-term fellowship. B.G. received a predoctoral FPU training grant from the Spanish Ministry of Science and Innovation. P.G. was supported by a long-term EMBO fellowship.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
From the time Patti greets you, thru Bernie's helpful hand at breakfast the next day, the stay at the Trinkle Mansion will be one you won't forget. We have stayed here several times and each time it seems to get better. Try it, you'll like it!
- Official Description (provided by the hotel):
- Just minutes off I-77 and I-81 in Wytheville, Virginia. Located halfway between Charleston WV and Charlotte NC on I-77 and between Briston TN and Roanoke VA on I-81. Perfect stopover for those traveling to Myrtle Beach, Charleston, Savannah or Florida. Restored historic Bed & Breakfast offers four elegant guest rooms plus a private cottage for tranquil retreat or romantic getaway. Queen-sized beds with fine linens and private bathrooms with spa-inspired amenities. Delicious three-course breakfast. ... more less
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text:
Anyone, please, i need help installing plugins
home to us
Anyone, please, i need help installing plugins
home to us
Try reviewing Managing_Plugins.
thanks for reply, but i don't even know how to get them on the site
when i unzip the plugin and upload to plugins they do not show up
Please try reading the page at the link I posted above.
thanks, i will
not working for me, it sends me back to plugins saying that no plugin was found
i am trying to install this
I'm sorry but these forums do not support commercial products. Only the free plugins downloaded from http://wordpress.org/plugins/. Please contact the plugin's vendor directly with any questions about commercial products.
Where does it say that is a plugin?
[Moderator Note: Please do not post in uppercase & shout at us. Post de-capped.]
i downloaded it from plugins, so i assumed that it was one
That script has nothing to do with wordpress.org
sorry but it's a habit, i usually type only in upper case, thanks for your help, but my last message was not directed to you
This topic has been closed to new replies.


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New Adult Asian Materials
New arrivals added to our Adult Asian Materials in the last 7 days
|Date added
|Sansho the bailiff = SanshoÌ dayuÌ by
|Jul 27, 2016
|Sayonara [Japan version] by Sakerock (Musical group), performer.
|Jul 27, 2016
|Scandal show by Scandal (Musical group : Japan)
|Jul 27, 2016
|Second half by
An Indie charmer meets an struggling film producer who he suspects that she could be his daughter. Before he could tell her his hunch, she reveals an shocking confession.
|Jul 27, 2016
|Star Tanjou [Japan version] by Challenge (Musical group), performer.
|Jul 27, 2016
|Take off = Wu ge tiao xue de shao nian by
|Jul 27, 2016
|Taking of Tiger Mountain = Zhi qu Weihu Shan by
Ka-fai is a ruthless bandit stealing and ruling land from his fortress atop Tiger Mountain. Gengxin captain of the People's Liberation Army launches attacks on the mountain fortress to overthrow the dictator.
|Jul 27, 2016
|Ten (Normal edition) (Japan version) by Orange Range (Musical group : Japan)
|Jul 27, 2016
|Ten thousand spirits = Mansin by
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As friends discuss bizarre events about their teacher's life, each new story told changes the meaning of the previous.
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Comic-Con Trailers: The Best of the Best, Ranked
Film, TV
Showing results 41-80 of 176
"Alpha Flight" co-writer Fred Van Lente talks to CBR TV about mixing political intrigue with super heroes and touches on copyright and creator's rights via "Comic Book Comics."
In July's "Daredevil" #1 by writer Mark Waid and artists Paolo Rivera and Marcos Martin, Matt Murdock returns home to resume his career as a super hero and a lawyer. CBR spoke with Waid about the series.
Marvel has released a teaser image for the all-new "Punisher" ongoing series promising the announcement of a brand new creative team at the helm. Marvel plans to reveal more during C2E2.
"Bone" and "Rasl" creator Jeff Smith spoke with CBR TV about the recent controversy over his beloved all-ages series, the upcoming "Bone" novel and other books, the origins of "Rasl," its ties to Nikola Tesla and more.
Writer JT Krul spoke with CBR TV about his future plans with Aspen, living up to Michael Turner's legacy, the change in direction he's currently helming on DC Comics' "Green Arrow," his time working on "Seinfeld" and more.
Hellboy creator Mike Mignola spoke with CBR TV about the growing challenge of keeping the Hellboy Universe continuity straight, Duncan Fegredo's Hellboy future, Mignola's return to Hellboy interior art and more.
Writer/artist Jonathan Hickman spoke with CBR TV about his career's meteoric rise, his work on the critically acclaimed debut issue of "S.H.I.E.L.D.," his run on "Fantastic Four," what's coming up in "Secret Warriors" and more.
DC Comics Chief Creative Officer Geoff Johns joined CBR TV at C2E2 to discuss his new position, Aquaman as an A-List character, working with Peter Tomasi, his drive to grow as a creator, "Brightest Day" and more.
Chicago Bears linebacker and lifelong comic book fan Lance Briggs joined CBR TV during the inaugural C2E2 show to discuss his love of the medium, ethnic diversity in comics, his involvement with Top Cow and much more.
Jeph Loeb spoke with CBR TV at the recent C2E2 convention about keeping the identity of Red Hulk a secret, working on the mystery with Ed McGuinness, his Ultimate Universe titles, Arthur Adams, Frank Cho and much more.
What's up with "Runaways?" How about Iron Fist and Norman Osborn? Marvel's philosophy about digital distribution? Joe Quesada answers your questions and more in CBR TV's exclusive CUP O' VIDEO Q&A!
The pre-con meeting of retailers and publishers held of new series and trades from Marvel and DC and the unprecedented idea of moving new comics day to Tuesday. CBR has an in-depth report from the conference.
Mark Waid spoke with CBR TV during C2E2 about the new "Irredeemable" cologne, the BOOM! Studios title's one year anniversary, upcoming plans for the series, how the industry has changed in recent years and more.
In front of a sold-out auditorium, Neil Gaiman read his short stories and poems, answered fan questions, and told not a few amusing anecdotes Saturday night at the C2E2 benefit for the Comic Book Legal Defense Fund
BOOM! Studios Editor-in-Chief Mark Waid and acclaimed "Bone" and "RASL" creator Jeff Smith fielded questions from the audience and provided lots of laughs and memories in a Sunday panel focusing on independent comics.
Top Cow's C2E2 panel covered a wide range of topics including the company's road to "Artifacts," the jaw-dropping art of "Velocity" and a special guest appearance from Chicago Bears linebacker Lance Briggs.
"Turok, Son of Stone" will be the third classic Gold Key character reinvented by Jim Shooter, Dark Horse announced at C2E2 last weekend. CBR spoke with Shooter about his latest take on the time-tossed adventurer.
Joe Benitez's new series "Lady Mechanika" and big promises for digital publishing in the near future were topics of discussion at Aspen's panel, where they also confirmed statuses on the "Fathom" and "Soulfire" films.
The show organizer takes CBR News on a wrap up of the con from attendance to the scale of the show and from awareness to the future dates and location of the Chicago Comics & Entertainment Expo.
"Fables," "Angel" and "JSA" writer Bill Willingham pledged one spoiler per project during his C2E2 panel and subsequently revealed to fans a marriage in store for Snow White, new prophecies for Spike and more.
Evan Dorkin, Jill Thompson, & Scott Allie spoke exclusively with CBR about "Beasts of Burden/Hellboy," in which Mike Mignola's signature creation teams with the four-legged detectives of the supernatural.
DC Comics co-publishers Dan DiDio and Jim Lee entertained fans in an informal panel, basking in enthusiasm generated by "Blackest Night," taking their lumps for "Amazons Attack" and announcing a new miniseries or two.
Mike Mignola arrived at his C2E2 panel, declaring that he was there for the audience and spending the hour-long session discussing Hellboy, answering questions and hinting at things to come in the Hellboy universe.
In the wake of the high-profile debut for the Chicago Comics & Entertainment Expo, CBR News catches up with responses from major players from DC and Marvel to Dark Horse and Image and more.
Jim Shooter, Dennis Calero, Bill Reinhold and Chris Warner took to the stage during C2E2 to chat with fans about the imminent re-launch of "Doctor Solar," "Magnus, Robot Fighter" and "Turok" at Dark Horse.
The men behind the Wildstorm Universe caught readers up on current events in the Wildstorm U and announced Tony Harris' next project post-"Ex Machina" and new crossover book with IDW "X-Files/30 Days of Night."
From a brand-new "Farscape" threat to the return of Cthulu to a line of family-friendly Stan Lee created titles, BOOM! Studios treated C2E2 attendees to news, previews and more than a tease or two. CBR News was there.
At Chicago's C2E2, Archaia discussed their present and future in the digital world of comics and announced an all new brand extension banner. Black Label, specifically dedicated to Hollywood partnership titles.
Archives: All | Comic Books | TV/Film | Video Games | Web Comics


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Release date: May, 2010 * Movie Release
Studio: Hustler Video - Director: Will Ryder - Serie: Barely Legal Innocence
Duration: 24 mins, 35 secs (aprox) - Movie Segment: 00:02:35 - 00:26:10
Starring: Allyssa Hall, Johnny Nitro Search for more content with them
Story: Allyssa Hall is exactly what Johnny Nitro needs. Allyssa sucks on it like she was licking her first ever cock and her fresh tongue was uncertain of exactly how to do it. Once she started to get her pussy fucked, she relaxed and totally opened up allowing him to plow his meat deep inside her body giving her a new experience before splashing down all over her face with a load of population chowder.
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As a rustic decoration lover, I adore twig vases for floral arrangements. These twig vases are not only perfect for wedding centerpieces but can be used as favors or decorations for your home. Twig vase centerpieces are versatile in the way that you can use any type of container you have at hand, from old pickle jars to coffee cans and only need to stick twigs of equal length to the container using glue gun or rubber bands. Martha Stewart has a detailed guide on how to make twig vases. To dress up these twig vases, tie them with ribbons, raffia or lace.
{via Martha Stewart}
{via Martha Stewart}
{via Nini Makes}You Might Also Like:
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Top Definition
The act of shoving an apple down the back of someone's shirt, causing them to have an apple humped back.
Ahh man, I can't believe you just apple-backed me again!
de Golden Delicious The Second 08 Ianuarie 2009
Trending Now
E-mailurile sunt trimise de la email@example.com. Nu-ți vom trimite mesaje nedorite.×


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Nello Luchi
User Stats
Profile Images
User Bio
Janell Smith is considered by many to be one of the most impressionable young artist of the new millennium. With a street buzz that is currently getting bigger, she does it all with a gracefulness and ease. With her punch lines, amazing lyrical techniques, and gift for entertaining, Janell is unquestionably a threat to the music world. Unlike most artist and urban music's rap stars. Janell Casaundra Smith also known as Nello Luchi was born to do this. She writes her own songs as well as performs them, and her creative wit is definitely up to par with her stylish demeanor. Although sexuality is apart of Janell's image, it isn't as important to her as say Foxy Brown, or Lil Kim, it is mostly based on her lyrical skills, which is never in doubt. Janell was born in no other the City of New York, borough of Brooklyn. She originally started rapping at the age of 11. While some artist rap to become part of the in crowd or to be down, Nello started rapping because of her love for poetry. She also loved hip hop music and began combining the two into something of her own. Janell's inspiration comes from artist such as Jay-z, Notorious B.I.G., Slick Rick, A Tribe Called Quest, Mc Lyte, Lil' Kim, Run DMC, Wu-Tang, as well as many artist of today. Nello brings a youthful, soulful, rock and urban sound/style to the game. Like other artist, Janell uses music to express what she feels and also to have fun. Nello is a very determined person and also a perfectionist. She creates her own hooks for her songs and although she isn't a singer by heart, she executes them to perfection. There is none other like her; she is without a shadow of a doubt bringing something new, fresh and indisputable to the table. Its definitely going to be hard being a female artist in the game, next to the Realest Chick Out. Ms. Janell Casaundra Smith a.k.a Nello Luchi


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TomTom HD Traffic boosted by capital injection
TomTom has done a deal with Transport for London to get more detailed London traffic info into its HD Traffic service.
TFL will provide TomTom with its traffic data - so TomTomers will have access to information such as where road works are taking place, where accidents have happened, temporary road closures (handy come Olympic-time) and where traffic is likely to be disrupted.
They'll also get the severity of the delays thanks to TFL's 360 mile red route network in London and information fed into its control centre by London's 7,500 buses, event organisers and road works contractors combined with speed data tracked from connected TomTom devices and Vodafone mobile phone handsets.
"It's been proven that stress caused by driving can be hazardous to your health" says Helene Raad, traffic business development manager at TomTom.
"With the addition of Transport for London's data in to the latest version of TomTom HD Traffic we can provide even more detailed traffic incident reports helping you to steer clear of traffic jams, and avoid the accompanying negative side effects to your health."
TomTom recently launched version 5.0 of the HD Traffic software, adding information on automatic road closures to pinpoint the exact section of the route that's disrupted as well as improved standstill detection.
The TFL update should be beamed over to TomTom HD Traffic subscribers automatically for free. Potential new users can head over to www.tomtom.com/services to take a look at the various Live packages on offer.
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Motorcycles > USA > FL > Winter Haven Area > Custom
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Red Custom Motorcycles for Sale in Auburndale, Florida(1 - 4 of 4)
2015 Harley-Davidson® XG500 Street⢠500Harley-Davidson · Red · Saint Petersburg, FLSTART RIDING TODAY! 2015 Harley-Davidson® Street™ 500 You've got 500cc of liquid-cooled Harley-Davidson® V-twin engine and attitude ...Marketplace User ·1 week ago on ChopperExchange
2015 Harley-Davidson® FLHTKSE CVO⢠LimitedHarley-Davidson · Red · Palm Bay, FLPlease call and ask for Paul: 888-658-XXXX 2015 Harley-Davidson® CVO™ Limited We've built grand American touring from the ground up. ...Marketplace User ·4 weeks ago on ChopperExchange
$26,999 2016 Polaris Slingshot Reverse Trike SL Red PearlPolaris · Red · Kissimmee, FL… , The most amazing ride on the road today with a Roof and 2.99% for 60 mnths with approv...Kissimmee Motorsports ·Over 4 weeks ago on Cycle TraderTools
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2012 Harley-Davidson® XL1200C Sportster® 1200 CustomHarley-Davidson · Red · Leesburg, FLMany leasing and financing options available. Come check us out today! Open seven days a week! 2012 Harley-Davidson® Sportster® 1200 Cu...Marketplace User ·Over 4 weeks ago on ChopperExchangeTools
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EEE meeting to be held in BrandonJuly 05,2013EEE meeting
next week
BRANDON Two state agencies are holding a public forum on their plans for the Eastern equine encephalitis (EEE) virus next week.
The state Department of Health and Agency of Agriculture, Food and Markets will hold the focum on its 2013 Arboviral Surveillance & Response Plan from 5-7 p.m. July 10 at the Brandon Town Hall.
The plan includes the trapping and testing of mosquitoes, human and animal illness surveillance, public information, educational outreach and action that may be taken in response to signs of increase risk for illness.
In 2012, EEE was detected in mosquitoes for the first time in Vermont. Two people died from the disease.
For more information, visit healthvermont.gov or call 1-800-913-1139.MORE IN This Just In
MIDDLEBURY — Middlebury Police arrested Ronald Bean, 52, on two counts of... Full StoryMore than 26,000 people were without power when severe thunderstorms rolled through the state in... Full Story A Rutland man was allegedly drunk and playing Pokémon Go when he crashed his... Full Story
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Volcanic Ash Cloud AlertPosted on 20 August 2014
Over the last 2-3 days, the Icelandic volcano Bardarbunga moved from Green alert status, to Yellow, then to Orange. If the alert moves to Red, an eruption is imminent or has started, with potential massive impact on the aviation industry. Two eruptions in Iceland in 2010 and 2011 caused over 100,000 flights over Europe to be cancelled with an economic impact in excess of €5 billion.
Lack of sophistication in ash cloud forecasting, along with shortcomings in the detection and quantification of the ash dispersion, led to blanket no-fly decisions and flight cancelations. NUI Galway's Centre for Climate & Air Pollution Studies (C-CAPS) and ICHEC have deployed a rapid-reaction ash forecasting system that relies on algorithms developed by C-CAPS, ICHEC's Fionn supercomputer and data provided by Met Éireann. The current simulations provide a hypothetical scenario that can be used by the aviation authorities and airlines to plan for 1-4 days into the future in case of an eruption. In short, aviation stakeholders are now significantly more informed of potential ash dispersion patterns prior to and during any eruption.
The ash cloud forecasts can be found on www.macehead.org
|ICHEC's own Ruairí Short to compete in World Orienteering Championships
Ruairí Short will compete this August at the World Orienteering Championships in Sweden
|Four positions open in ICHEC
A range of computational research positions are currently available at ICHEC.
|ICHEC secures Intel funding to predict extreme wave events
The Irish Centre for High-End Computing (ICHEC) partner with Intel and world-leading US institute NOAA on modernizing operational wave forecasting
|The subtle power of high-performance computing
ICHEC's director JC Desplat featured in the Irish Times and Silicon Republic
|SESAME Net SME online survey
SESAME Net SME online survey
|ICHEC presents at IXPUG Workshop in Czech Republic
ICHEC presents progress on recent work on Intel Xeon Phi at Intel Xeon Phi User Group (IXPUG) Workshop in Czech Republic
|PRACE CodeVault Launched
Open repository of HPC code samples launched
|Code Modernization Workshop & 2-Days Hands-on Seminar on Preparing your code for Knights Landing
ICHEC, Intel and Bayncore hosting 3-days of Intel Xeon Phi activity
|SESAME NET - Connecting Irish SMEs with HPC
ICHEC is co-leading H2020 project with HPCWales
|Powering Public Sector Innovation with ICHEC and High-Performance Computing
Big Data brings big benefits
|International Big Data benefits unlocked by Irish led innovation
Ireland bridging the gap between high-performance computing and official statistics
|Irish Climate Change-what is in store?
ICHEC publishes Irish climate change report
|ICHEC collaborates with Colfax International
ICHEC collaborates with Colfax International on preparing weather code for Xeon Phi


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Smith Moore Leatherwood Honors Attorney For Pro Bono Work In 2012
Wednesday, January 30, 2013
- Organization: Smith Moore Law
- Link: http://www.smithmoorelaw.com
James Mason is an associate attorney in Dorsey & Whitney LLP's Minneapolis office. He volunteers with Volunteer Lawyers Network (VLN) in its criminal expungement workshop. At the workshops, volunteer attorneys like James help people complete the lengthy and detailed court pleadings required for expungement using the HotDocs assembly tool hosted on ProJusticeMN.org. READ MORE
Legal info for the public
LawHelp.org Named Best Law Site in 2007 Webby Awards


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Wasn't a phablet.
Instead of educating the public about not swallowing random crap, researchers at the school want people to swallow a new folding origami robot they've developed.
Somebody call Scully and Mulder.
It isn't over until the X-ray sings.
A newly developed CT scan technique means that an animal can be awake and standing up while it's being imaged.
That's exactly what a company called Mobility Designed has done.
It takes the modified scalpel just 400 milliseconds to distinguish between healthy brain tissue and the cancerous stuff using a very clever test.
It's basically a way to help people keep track of their medical treatment, and to share information with doctors remotely.
He's been suffering for a decade.
App connectivity makes it to the baby-making world.
It's not a new idea, but it's the first method that does the cooling automatically.
This new limb, which could help the man play with two hands again, is a collaboration called The Phantom Limb Project. See what they did there?
It sounds like something straight out of a comic book, but after losing his sternum and part of his rib cage to cancer, a 54-year-old Spanish man received the world's first 3D-printed chest prosthetic made from titanium.
GE has developed new imaging software that, instead of using harmful X-rays, relies on ultrasound, which is safe for even foetuses. With it they can generate real-time 3D views of our internal organs, like this one of a beating heart. Read more >>
Donations are down - so it is your patriotic duty to help reverse this trend.