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A filmmaker took to 30-year-old disaster site Pripyat, Chernobyl equipped with a camera, a drone and a GoPro.
Apple's latest ad, in which Justin Timberlake and Jimmy Fallon behave as you might expect gamers to, causes online ructions among some gaming devotees.
Just in time for for Thanksgiving, Old Spice shows what happens when both parents stalk their sons. Can this ad achieve the fragrant heights of its predecessor?
Some traditions will not die. Wresting a home theater system out of someone else's hands, for example.
Enlisting actor Dave Franco to his cause, Conan O'Brien tries to strut his online dating stuff. It's painful.
Whether you serve a nerdy turkey for Thanksgiving or a gingerbread AT-AT for Christmas, you've got plenty of options for boasting your geek pride at the holiday table.
A 150-second extravaganza from Samsung shows a man trying to impress a woman with, yes, his new gadget. What follows is the stuff of Hollywood. Sort of.
A Google Glass developer with a clear vision of what Glass can be, Brandyn White sees how Glass can be a force for good with a feature that Glass doesn't even have yet.
The inventor of the Google Glass Sunshade says his motivation was to make it easier to see the Glass graphics when in sunshine. Coincidentally, it also covers up the light that tells others the glasses are in use.
Enjoy the lush, plush fur of a flattened Wookiee beneath your feet when you deck your living room with a Chewbacca rug.


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Find records of Mabel Arinto
Find birth, death, marriage, military, and other records of Mabel Arinto at Ancestry.com.Search for records
Biography & Family History Add details
Mabel Arinto (1898 - 1981) was born on November 22, 1898. She was born into the Arinto family.
Mabel had no relationships. Mabel died in January 1981 at 82 years of age.
Mabel Arinto's last known residence is at Staten Island, Richmond County, New York.
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subset: commoncrawl


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On eve of trade deadline, Brodeur and Devils top Red Wings
If this does happen to be the way Martin Brodeur's career as a Devil ends, then at least he leaves on a high note. On the eve of the trade...
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The In-Cider Scoop: Sidewood Launches New Cider
Sidewood held a VIP launch recently, treating guests to an intimate sneak peek of the 2014 award winning 'Champion South Australian Cider'.
Sidewood held a VIP launch recently, treating guests to an intimate sneak peek of the 2014 award winning 'Champion South Australian Cider'.
The World Premiere of Unbroken was held last night in Sydney and was attended by Director and Producer Angelina Jolie and the stars of the film Jack O'Connell and Miyavi. Check out all the photos here.
The Variety Vintage Luncheon was held at The National Wine Centre on Friday 14 November.
Presented by Asahi Premium Beverages, the Asahi Super Dry Silver Sessions hit South Australia at The Lion Hotel last Thursday, November 13th.
Adelaide Fashion Designer, Christina Tridente, held her first ever parade for couture+love+madness. Here are some of the photos from the evening.
Australian Dance Theatre presented the opening night of their return season of Proximity at the Dunstan Playhouse, which also marked the start of the 50th anniversary celebrations in 2015.
After months of anticipation, Uber Adelaide had their official launch last night at Jolley's Boathouse.
Photos from Mariah Carey's The Elusive Chanteuse World Tour at Adelaide Entertainment Centre
Coopers recently hosted a special event to celebrate Glenn Coopers recent retirement.
The Westminster School Foundation hosted its inaugural Long Lunch on the Green at Westminster School on Sunday (26 October).


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Features / Film
Head to Head: Chadwick Boseman and Tate Taylor
Published on 18 November 2014
Ahead of the release of the James Brown biopic Get On Up director Tate Taylor and actor Chadwick Boseman go head to head.
Meet the Model: Edward Wilding
Published on 12 November 2014
Get to know Ed Wilding, the Mancunian mega model who has won over the fashion industry with his Adonis looks, humility and great sense of humour.
Fashion / Features
Hungry: Soo Joo Park
Published on 10 November 2014
Soo Joo Park tells us why blondes have more fun.
One From The Archives: Aneurin Barnard
Published on 09 November 2014
The valley boy who is proving to be one of our most promising acting talents.
Features / Film
What is a Sci-Fi?
Published on 09 November 2014
What makes a film science-fiction? Hunger debates what has happened to the sci-fi genre.
Film / Features
One From The Archives: Mark Strong
Published on 01 November 2014
Choosing the right part is like choosing the right coat, says actor Mark Strong. It's all about the fit.
Film / Features
One From The Archives: Olivia Colman
Published on 26 October 2014
The BAFTA-winning actress knows all there is to know about the crying game.
Features / Film
Top Ten Films from the London Film Festival
Published on 20 October 2014
As the BFI London Film Festival draws to a close we round up the top ten films from this year's festival.
Features / Film
The Interview: David Dobkin – The Judge
Published on 18 October 2014
"I stay emotionally attached because I want to know if certain buttons are getting pushed the right way," director David Dobkin tells us about his personal project and latest film, The Judge.
Features / Film
One From The Archives: I Dare You: Jamie Campbell Bower
Published on 18 October 2014 53 Comments
Remember when we played a game of dares with Jamie Campbell Bower? And remember what he did…


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Mud mixing systems from KEMTRON, TT Technologies, Triflo Intl., American Augers, Ditch Witch, Vermeer and Mud Technology Intl.
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Backhoe loaders from Case, Volvo, Caterpillar, Bradco/Paladin Construction Group, Kubota Tractor and Terex.
Copyright Oildom Publishing Company of Texas, Inc. All rights reserved.


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Oh, wait, a few of you are interested in something else that happened last night…that's right, there was an election. Before contemplating what this means for the nation, let's quickly check my predictions.
Now let's think about the consequences for America.
NEW TIME Today, at 9:30 AM PT: Get the Market Movements in Advance; Williams Edge Webinar for November 26th, 2014 | John Ransom


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'In Champions League T20, most teams want to beat Mumbai Indians'Mumbai Indians captain Kieron Pollard celebrates with teammates the fall of a Northern Knights wicket during the qualifier of the Champions League Twenty20 in Raipur. Photograph: BCCI Mumbai Indians skipper Kieron Pollard rued the fact that batting let his team down as the defending champions crashed out in the qualifying round of the Champions League T20 in Raipur on Tuesday. "They (Northern Knights) fielded well as we could have scored at least 150-160. What happens in ... Rediff.com, 2 months ago
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Huntington Local
Community news from Asharoken to West Hills and every place in between.
BloggersMary Beth Foley Lida Russo Mike Earley Tim Healy
ArchivesMore »
Police ask help on suspect in South Huntington credit card thefts
Police are asking the public's help in identifying a woman who took credit cards from pocket books in a South Huntington Hallmark.
Police say the woman, seen above in video surveillance photos, took the credit cards and other items at the store on Route 110 on Wednesday, June 17, at about 3 p.m. She is described as a white female, 30-40 years old, with long, dark hair. She was wearing jeans and sunglasses.Anyone with information about this crime is asked to call Crime Stoppers at 1-800-220-TIPS. Calls will be kept confidential and the caller will be eligible for a cash reward of up to $5,000 for information that leads to an arrest.
Have you got a story? We want to hear from YOU. E-mail us a story -- even a photo or two -- about what's going on in your community.
Tags: South Huntington , crime


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Aug 11
Posts: 3820
babyrocket says:
me old chitty lilo good to talk to you today, all being well i will see you sunday roughly around 11.30 ish, along with a few other old faces, have i to bring my boxing gloves just in case any one wants to have a scuffle.?
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me up that little conflab, after a very piss poor last few weeks.
Dec 10
Posts: 12075
preunit says:
rocket man, gunga and meself are planning to arrive at 11.00ishski, hopefully meeting up with one or two along the way.
Jun 12
Posts: 3672
calamityjane says:
Probably more like a tea dance and a soft (clarks) shoe shuffle than a scuffle. Take ear plugs for the chas and dave ensemble and a sick bag for the jellied eels.
[This Reply has been modified by the Author]
but not got a pair of clarks shoes to ny name.
no need for the reefs, good old alpinestars for me, and they aint that heavy if i need to leg it, as for this past few weeks its been bloody awful, but shit like that only makes you realise just how sweet life truly is, hence the softening up i hopefully have very few enemies and any one i have offended i can only re iterate my apologies.
Feb 11
Posts: 2652
gungerdin8 says:
me old bobby dazzler good to chat to u also dude
right up 4 the ponderosa
be good to put te world2 rights dude
oh n don't worry I got ya back !!!!!!!!!!!!!!!!!!!!!!!
lets av it !!!!!!!!
sunday then, just hope mr unit has got the weather right, nowt worse than a soggy wet arse and a reet long ride home.
Adopted a geordie accent, and i'm wearing a dress!!
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WEEKEND BEST BET: American Craft Show Sarasota
Date: December 2, 2010
by: Heidi Kurpiela | A&E Editor
Is the mall too crowded and mass-market for your tastes?
Do independent artists a favor this weekend and patronize The 18th Annual American Craft Show Sarasota.
Scheduled for Dec. 3 to Dec. 5, the show features work by more than 90 of the nation's hottest artists, including local ceramic artist Scott Causey, a Ringling College of Art and Design graduate.
The three-day craft spectacular, which is held at the Sarasota-Bradenton International Convention Center every year, isn't your run-of-the-mill craft fair.
Sponsored by the American Craft Council and managed by Richard Rothbard, a New York-based artist and gallery owner, the event is among one of the top retail craft conventions in the country with pieces priced from $50 to $1,000.
Tickets are $9 per day for regular admittance. Senior tickets are $8 and student tickets are $5. Children under 10 are free.
The show is open from 10 a.m. to 5 p.m. Friday, Dec. 3 and Saturday, Dec. 4; and 10 a.m. to 4 p.m. Sunday, Dec. 5.
The Sarasota-Bradenton International Convention Center is located at 8005 15th St. E., Sarasota.
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- Nov
28 The Jazz Club of Sarasota Presents "Jazz at Two" with Alex and Judi Glover
2:00 pm - 4:00 pm
- Nov
29 Dickey Betts & Great Southern Charity Concert
6:00 pm
- Nov
30 St. Andrews Day Celebration
3:00 pm - 4:30 pm
- Dec
2 Perlman Music Program/Suncoast Free Ticket Distribution at USFSM Rotunda for Winter Residency Musical Events
10:00 am - 2:00 pm
PHOTO GALLERY: Bird Key Yacht Club's Commodore's Ball
On Nov. 22, Bird Key Yacht Club held its annual Commodore's Ball.
PHOTO GALLERY: St. Mary, Star of the Sea, Christmas Bazaar
On Nov. 22, St. Mary, Star of the Sea, Catholic Church held its annual Christmas Bazaar.
PHOTO GALLERY: Longboat Key Chamber of Commerce Chairman's Reception
On Nov. 20, the Longboat Key Chamber of Commerce held a Chairman's Reception at Amore by Andrea, which recognized its former chairs from 1993 to 2013.


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I am looking for information about John W. Hunter and/or his wife Mary Ann Hern Hunter. They moved to Gilpin Co. from Andrew Co., MO in the early 1860's. Supposedly John died around 1865. Any information you might have would be greatly appreciated.
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Plenty MoreHardback Ebury Press
List price $42.30
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- Publisher: Ebury Press
- Format: Hardback | 288 pages
- Dimensions: 198mm x 276mm x 32mm | 1,509g
- Publication date: 11 September 2014
- Publication City/Country: London
- Sales rank: 7
Product description
Yotam Ottolenghi's Plenty changed the way people cook and eat. Its focus on vegetable dishes, with the emphasis on flavour, original spicing and freshness of ingredients, caused a revolution not just in this country, but the world over. Plenty More picks up where Plenty left off, with 120 more dazzling vegetable-based dishes, this time organised by cooking method. Grilled, baked, simmered, cracked, braised or raw, the range of recipe ideas is stunning. With recipes including Alfonso mango and curried chickpea salad, Membrillo and stilton quiche, Buttermilk-crusted okra, Candy beetroot with lentils, Seaweed, ginger and carrot salad, and even desserts such as Roasted rhubarb with sweet labneh and Quince poached in pomegranate juice, this is the cookbook that everyone has been waiting for.
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Author information
Yotam Ottolenghi completed a Masters degree in philosophy and literature whilst working on the news desk of an Israeli daily, before coming to London in 1997. He started as an assistant pastry chef at the Capital and then worked at Kensington Place, Launceston Place, Maison Blanc and Baker and Spice, before starting his own eponymous group of restaurants/food shops, with branches in Notting Hill, Islington and Belgravia. He opened the restaurant NOPI in Piccadilly in 2011. Yotam is the author of the bestselling Ottolenghi: The Cookbook, Plenty and Jerusalem. He has presented two series of Ottolenghi's Mediterranean Feast on More 4 and Channel 4. Plenty More is his fourth book.
Review quote
"Its this approachability that is the books greatest strength; it gives inspiration, as well as just great recipes, and it's not just for vegetarians." Time out


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Internet kiosk public access PC software to restrict access to system; allow only certain applications and features such as Internet Explorer. Limit access to the PC desktop using authentication by username and password, time-locked serial number to allow user to access PC for a given period of time only or by a combination of both. The best solution for Internet cafes, public libraries, information terminals, home PCs to limit childrens
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Deskman allows you to protect desktops easily. Restrict access to system settings and keep your desktop tightly secured from a simple interface. Home users need a simple way of protecting their computers on coffee breaks, or restraining access to their desktop on shared computer environments. Securing your Desktop has never been so easy. Free for personal use.
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Folder Guard Professional 7.5
Folder Guard Professional for Windows XP/2000/Me/98 - Powerful security and access control utility that lets you hide or password-protect files and folders, restrict access to Control Panel, and more. Makes folders invisible to any application, including Explorer, Office, command prompt. Highly customizable and user friendly. For personal systems or corporate networks. Evaluation version is fully functional. http://www.winability.com/
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Folder Guard XP Edition 7.5
Folder Guard XP Edition for Windows XP/2000/2003/2005 - Powerful security and access control utility that lets you hide or password-protect files and folders, restrict access to Control Panel, and more. Makes folders invisible to any application, including Explorer, Office, command prompt. Highly customizable and user friendly. For personal systems or corporate networks. Evaluation version is fully functional. http://www.winability.com/
NetSupport Manager - PC Remote Control 9.0.0
Perform remote support and management on multiple systems simultaneously over a LAN, WAN and the Internet with this PC remote control software. Supports mobile users effortlesly and also remote sites and servers. Provides speedy, secure remote PC access, remote deployment, a hardware and software inventory, centralised management, file transfer, remote helpdesk, support for Pocket PC and Linux, a new whiteboard feature and record and replay.
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1st Network Admin 1.61
Comprehensive protection for Windows-based network workstations under a public environment. It lets you restrict access to every corner of PC workstation, apply executable patches remotely, and schedule reboots, shutdowns, and Windows Explorer restarts from a single administrator's computer connected to a TCP/IP-based network or Internet. In total, 1st Network Admin supports over 600 different security restrictions, options and tweaks.
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PC Security Tweaker 6.5
PC Security tweaking software to tweak Windows-based computers. Tweak access to lots of Control Panel applet functions, enforce users to use separate system profile folders, disable selected Start Menu items, lock and protect disk drives in My Computer, disable the DOS and command prompt, interruption boot keys, real DOS mode, Registry editing, taskbar, task manager, and network access, hide desktop icons, and much more.
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Free Remote Desktop software. With t-VNC you can control remote computer viewing its Desktop and controlling by the keyboard and mouse. Includes advanced features, like Stretched view, Block remote computer and other. Easy in use.
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TrafficRefine 2.01
As a computer user you need to protect your system from malicious software and as a parent you evidently want the Internet to be as safe as possible for your child. TrafficRefine is a personal network filter which is used to restrict access to particular Internet resources from a local computer. The program was developed with parental control in mind and combines necessary functionality with extreme simplicity of use.
See also: traffic, control, parental, internet, access, limit, firewall, child, children, kid
LanAudit Remote Control 2.84
With PC-Monitor you can remotely control any computer as if you were sitting right in front of it - even through firewalls.Allows the Network Administrator to view and control network computers, having the remote desktop on the screen of his own PC and controlling that remote PC by using the keyboard and mouse of their own.
See also: Hardware Inventory, software Inventory, Data Loss Prevention, Document Operations, Stop Data leakage, Application Control, Web Access Control, Employee Computer Monitoring, Employee Monitoring, Computer Monitoring, Remote Control, Remote Install
Time Lock 126.96.36.199
Time Lock is a simple program, which allows to protect your computer against unwanted use. When the computer is locked by the Time Lock software, nobody can access documents on your computer, run programs, browse on the internet, etc. Time Lock is very often used by parents as a "child lock" for home PC. Simply let your child to play games or browse on the internet only for a certain time period.
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Library Barcodes Generator 126.96.36.199
Cost effective Library Barcodes Generator software from www.bulkbarcode.com url is useful for making various book bar code labels to access publishing industry details. Book tags maker tool supports all graphical file formats like jpg, tiff, gif etc to add images on stickers, coupons.
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These days, everyone has many different passwords, to login to a messenger or an e-mail account, security codes for online banking, PINs for his telephone and so on. 1-abc.net Password Organizer allows you to organize all your passwords and login data, you only have to remember your master password. Additionally, it offers many useful functions concerning secure password management, a random password generator, own encryption engine, txt export..
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Press Release Distribution
April 02, 2014 News
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text:
What's The Difference Between Partly Sunny & Mostly Cloudy?
By Steve Pool
Most Popular
Your Photos
These images of the sunrise with Mt. Rainier were taken around 8am Monday November 24th. The pictures were taken with a Nikon D3000 DSLR with a 50-200mm telephoto lens.
Taken from my roof.
Isn't Nov. supposed to be our nastiest weather month? Seems OK to me...


text:
Dr. Robert G. Day, MD
Family Medicine, Glenrock Health Centre
- 925 W Birch St
Glenrock, WY 82637
- 2 Office Locations
- (307) 436-9206
Dr. Robert G. Day has been practicing medicine for 15 years. Dr. Day graduated from the University of Tennessee College of Medicine at Memphis in 1999. He practices medicine in Glenrock, WY and 1 other location and specializes in Family Medicine. Dr. Day speaks English and Spanish.
Gender: Male
Years in Practice: 15 Years
Languages Spoken: English, Spanish
Medical School: University of Tennessee College of Medicine at Memphis
Graduation Date: 1999
Hospital Affiliations: Not Available
Specialties: Family Medicine
*This physician has multiple office locations. See Appointments for details.


text:
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November 11, 2014
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October 15, 2014
About Author
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text:
Breeder of Cockapoo & Schnoodle puppies in New York. & South Carolina Specializing in, Miniature Cockapoos , Toy and Miniature Schnoodles. Cockapoos SC, Cockapoos NY,South Carolina,New York.
A Dog Will Love You Forever!
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Puppies. We are breeders of some of the finest Mini
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Our breeding's are limited to and include both Cockapoos and Schnoodle usually do not exceed 7-9 litters in a year,and sometime less, to assure you get a BarMor Puppy please join our wait list.
Located in The Beautiful Finger Lakes area of New York
And up state South Carolina.
Nursery News
Check out the Nursery
We are in Geneva, NY May - December
And Greenville, SC January - April
Our Father, which art in heaven, Hallowed be thy Name.Thy Kingdom come. Thy will be done in earth, As it is in heaven.Give us this day our daily bread.And forgive us our trespasses,As we forgive them that trespass against us. And lead us not into temptation, But deliver us from evil. For thine is the kingdom,The power, and the glory,For ever and ever Amen.
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Cockapoos,CT breeder, PA breeder, home-raised puppies, home raised puppies, training book for puppy pack. DNA Checks on all breeding dogs.
Cockapoo and Schnoodle Puppies available to go just before Christmas.


text:
The cDNA that encodes a glycoprotein receptor from the tobacco hornworm which binds a Bacillus thuringiensis toxin has been obtained and sequenced. The availability of this cDNA permits the retrieval of DNAs encoding homologous receptors in other insects and organisms as well as the design of assays for the cytotoxicity and binding affinity of potential pesticides and the development of methods to manipulate natural and/or introduced homologous receptors and, thus, to destroy target cells, tissues and/or organisms.
1. A polynucleotide in purified and isolated form which comprises a nucleotide sequence which encodes a receptor that specifically binds a Bacillus thuringiensis (BT) toxin wherein said receptor has the amino acid sequence of the receptor encoded by the insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713, or wherein said receptor is a naturally occurring receptor that is encoded by a nucleotide sequence that hybridizes at 40�-50� C. in 1�SSC, or equivalent conditions thereof, to the cDNA insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713.
2. The polynucleotide of claim 1 wherein said toxin is the cryIA(b) toxin of B. thuringiensis subsp. berliner.
3. The polynucleotide of claim 1 wherein said toxin is a modified form of BT toxin.
4. The polynucleotide of claim 1 wherein said receptor is the BT-R1 receptor of the tobacco hornworm Manduca sexta encoded by the insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713.
5. A recombinant expression system for expression of a nucleotide sequence encoding a receptor which specifically binds a BT toxin, wherein said receptor has the amino acid sequence of the receptor encoded by the insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713, or wherein said receptor is a naturally occurring receptor that is encoded by a nucleotide sequence that hybridizes at 40�-50� C. in 1�SSC, or equivalent conditions thereof, to the cDNA insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713,
which expression system comprises said encoding nucleotide sequence operably linked to a promoter and optionally one or more additional control sequences in host cells.
6. Recombinant host cells modified to contain the expression system of claim 5.
7. A method to produce a receptor which binds a BT toxin which method comprises culturing the cells of claim 6 under conditions wherein said receptor is produced; and
optionally recovering said receptor from the culture.
8. The method of claim 7 wherein said receptor is disposed at the surface of said cells.
9. The method of claim 8 further comprising a step wherein said cells having said receptor disposed at their surface are recovered from the culture for use in an assay.
10. The method of claim 8 further comprising a step wherein said receptor is recovered from said cells having said receptor disposed at their surface.
11. Cells expressing BT toxin receptor disposed at their surface prepared by the method of claim 9.
12. The cells of claim 11 wherein the receptor is a naturally occurring insect receptor that binds a BT toxin.
13. A receptor produced by the method of claim 7 wherein said receptor binds a BT toxin.
14. An isolated polynucleotide that encodes the BT-R1 receptor of the tobacco hornworm Manduca sexta encoded by the insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713.
15. The polynucleotide of claim 14 wherein said polynucleotide has the nucleotide sequence of the insert of the plasmid deposited at the American Type Culture Collection as ATCC 98713.
16. A purified and isolated polynucleotide which comprises a nucleotide sequence complementary to a nucleotide sequence encoding a receptor which binds to BT toxin wherein said receptor has the amino acid sequence of the receptor encoded by the insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713, or wherein said receptor is a naturally occurring receptor that is encoded by a nucleotide sequence that hybridizes at 40�-50� C. in 1�SSC, or equivalent conditions thereof, to the cDNA insert in the plasmid pBT-R1 deposited at the American Type Culture Collection as ATCC 98713.
Work resulting in the present invention was supported in part by Research Agreement 58-319R-3-011 from the Office of International Cooperation and Development, U.S.D.A. and by Cooperative Agreement 58-5410-1-135 from the Arthropod-Borne Animal Disease Laboratory, Agricultural Research Service, U.S.D.A. and by Grant HD-18702 from the National Institutes of Health. The U.S. government has certain rights in this invention.
The invention relates to receptors that bind toxins from Bacillus thuringiensis and thus to pesticides and pest resistance. More particularly, the invention concerns recombinantly produced receptors that bind BT toxin and to their use in assays for improved pesticides, as well as in mediation of cell and tissue destruction, dissociation, dispersion, cell-to-cell association, and changes in morphology.
It has long been recognized that the bacterium Bacillus thuringiensis (BT) produces bacteriocidal proteins that are toxic to a limited range of insects, mostly in the orders Lepidoptera, Coleoptera and Diptera. Advantage has been taken of these toxins in controlling pests, mostly by applying bacteria to plants or transforming plants themselves so that they generate the toxins by virtue of their transgenic character. The toxins themselves are glycoprotein products of the cry gene as described by Hofte, H. et al. Microbiol Rev (1989) 53:242. It has been established that the toxins function in the brush border of the insect midgut epithelial cells as described by Gill, S. S. et al. Annu Rev Entomol (1992) 37:615. Specific binding of BT toxins to midgut brush border membrane vesicles has been reported by Hofmann, C. et al. Proc Natl Acad Sci USA (1988) 85:7844; Van Rie, J. et al. Eur J Biochem (1989) 186:239; and Van Rie, J. et al. Appl Environ Microbiol (1990) 56:1378.
Presumably, the toxins generated by BT exert their effects by some kind of interaction with receptors in the midgut. The purification of a particular receptor from Manduca sexta was reported by the present inventors in an article by Vadlamudi, R. K. et al. J Biol Chem (1993) 268:12334. In this report, the receptor protein was isolated by immunoprecipitating toxin-binding protein complexes with toxin-specific antisera and separating the complexes by SDS-PAGE followed by electroelution. However, to date, there has been no structural information concerning any insect receptor which binds BT toxin, nor have, to applicants' knowledge, any genes encoding these receptors been recovered.
The invention provides recombinant materials for the production of BT toxin-binding receptors as well as methods to employ these materials to generate receptors for use in screening assays for candidate pesticides. Since the native cDNA sequence encoding this receptor, designated BT-R1, has been retrieved from the tobacco hornworm, encoding DNA for receptors in other species of insects, as well as in other organisms, which have homology to hornworm receptor can be obtained.
Thus, in one aspect, the invention is directed to a polynucleotide in purified and isolated form which comprises a nucleotide sequence encoding a receptor that binds a BT toxin and other ligands and which has the requisite homology to the BT-R1 protein.
In other aspects, the invention is directed to expression systems for nucleotide sequences encoding the receptor, to methods of producing the receptor recombinantly, to the receptor as thus produced, to antibodies specifically immunoreactive with the receptor, to assay methods useful for screening candidate pesticides, to antisense polynucleotides corresponding to the coding sequence, to methods of targeting tissues and/or cells using the binding characteristics of the receptor, and to methods of manipulating tissues and/or cells using the function of the receptor.
FIGS. 1A-1F (SEQ ID NO:1 and SEQ ID NO:2) show the nucleotide sequence and deduced amino acid sequence of cDNA encoding the BT-R1 protein from M. sexta.
FIG. 2A-2F (SEQ ID NO:2) show the cadherin motifs in the amino acid sequence of the BT-R1 protein from M. sexta.
The invention provides, for the first time, sequence information concerning receptors that bind BT toxins in insect midguts.
The BT-R1 cDNA clone retrieved as described in the examples below encodes a protein having an identical amino acid composition with that described for the native receptor. Furthermore, toxin-binding specificity and immunoreactivity are similar. The native 210 kD BT-R1 specifically recognizes cryIA(b) toxin of BT-berliner; a Kd value of 708 pM was obtained for the native protein.
The cryIA(b) toxin selectively kills M. sexta larvae with an LC50 of 7.5 ng/cm2 of diet surface. BT-R1 binds the toxin under both reducing and nonreducing conditions and protease treatment of intestinal BBMV vesicles prepared from M. sexta showed that a 50 kD fragment of the 210 kD receptor is sufficient for toxin binding. The 50 kD toxin-binding domain is extracellular since the intestinal BBMV vesicles are oriented predominantly right side out as reported by Haase, W. H. et al. Biochem J (1978) 172:57. This is consistent with the characteristics of the deduced amino acid sequence of the cDNA clone described below, as well as with the binding of toxin to the surface of intact BT-R1 transfected human embryonic 293 cells as described in Example 3.
Whereas a particular cDNA clone from the tobacco hornworm has been described as illustration, the availability of this sequence information permits retrieval of corresponding receptors responsive to BT and related toxins from other species. This is conveniently accomplished by using the cDNA obtained in the present invention as a probe for screening cDNA or genomic libraries under conditions of stringency which eliminate false positives and retrieve substantially only corresponding receptors with coding sequences that are homologous to the coding sequence for the receptor of the present invention. Thus, the BT-R1 protein itself and receptor proteins encoded by a nucleotide sequence homologous to the native nucleotide sequence encoding BT-R1 are provided by the invention. Alternatively, PCR-mediated cloning can be used; however, this method does not take advantage of the detailed and complete information that resides in the availability of the nucleotide sequence encoding the full-length receptor from M. sexta. Also, PCR-mediated cloning introduces errors in natural DNA sequences. Thus, by using the full-length cDNA as a probe under conditions of appropriate stringency, only nucleotide sequences encoding the corresponding receptors will be obtained. The standard hybridization conditions include hybridization with nonspecific DNA such as salmon DNA at 50� C. and washing at 45� C. To obtain corresponding receptors having the lowest detectable homology with the receptor from M. sexta, the cDNA probe is hybridized under conditions of low standard stringency (30�-37� C. and 4-6� SSC. More closely related corresponding receptors are obtained by hybridizing the cDNA probe under moderate standard stringency conditions (40�-50� C. in 1� SSC). A clone containing the cDNA insert for use as a probe was deposited at the American Type Cell Culture Collection as ATCC 98713.
The distribution of receptors of appropriate homology in the animal kingdom is believed to be fairly wide. Indeed, it is thought that higher organisms such as mammals, including primates, contain corresponding receptors which are homologous to BT-R1 but respond to modified forms of BT toxins. In addition, other parasites such as nematodes, both those that afflict plants and those that afflict animals, will contain corresponding receptors.
Although one of the advantages of the use of BT toxins as insecticides is its specificity for certain orders of insects, this specificity is believed to result from the particular structure of the BT toxin rather than the unavailability of a corresponding mechanism in other insect orders. Thus, modified forms of BT toxin would be effective with respect to insects which contain homologous but slightly different forms of the receptor from that of the BT-R1 protein illustrated below.
As used herein, "A receptor that specifically binds a BT toxin" refers to a receptor which is homologous to the BT-R1 protein illustrated herein and which binds to either BT toxins themselves or to BT toxins that are sufficiently modified so as to bind these receptors which provide the required homology to BT-R1.
The criteria for inclusion of a receptor in the present invention are the requirements that 1) it behave as a receptor--i.e., be capable of being displayed at the cell membrane; 2) it be sufficiently homologous to the BT-R1 receptor described herein that a nucleotide sequence encoding the protein hybridizes under the stringency conditions described above to the nucleotide sequence encoding BT-R1 as contained in the plasmid deposited at the American Type Culture Collection as ATCC 98713; and 3) when displayed on the surface of a cell, it is capable of binding a BT toxin or a modified form of BT toxin that exerts a cytotoxic effect either on the cell in which the receptor resides or in a tissue with which the cell is associated.
The structural characteristics of the "modified BT toxin" are defined by the functional property set forth above, but it may be convenient to design modified forms of BT toxin by conservative amino acid substitutions or other known protein-manipulating techniques applied to naturally occurring BT toxins.
The presence of similar receptors in noninsect organisms as well as other insects besides those harboring BT-R1 is supported by the sequence similarity of the BT-R1 protein to that of the various members of the cadherin superfamily of proteins, which are membrane glycoproteins believed to mediate calcium-dependent cell aggregation and sorting. See, for example, Takeichi, M. Science (1991) 251:1451; and Takeichi, M. N Rev Biochem (1990) 59:237.
Included in this superfamily are desmoglien, desmocollins, the Drosophila fat tumor suppressor, human intestinal peptide transport protein and T-cadherin. All of these proteins share common extracellular motifs although their cytoplasmic domains differ. Goodwin, L. et al. Biochem Biophys Res Commun (1990) 173:1224; Holton, J. L. et al. J Cell Sci (1990) 97:239; Bestal, D. J. J Cell Biol (1992) 119:451; Mahoney, P. A. et al. Cell (1991) 853; Dantzig, A. H. et al. Science (1994) 264:430; and Sano, K. et al. EMBO J (1993) 12:2249. Inclusion of BT-R1 in the cadherin superfamily is further supported by the report that EDTA decreases the binding of cryIA(b) toxin of BT to the 210 kD receptor of M. sexta (Martinez-Ramirez, A. C. et al. Biochm Biophys Res Commun (1994) 201:782Q).
It is noted below that the amino acid sequence of BT-R1 reveals that a calcium-binding motif is present. This is consistent with the possibility that cells having receptors to bind toxin may themselves survive although they render the tissues in which they are included permeable to solutes and thus effect disintegration of the tissue. Such a mechanism is proposed for the death of insects that ingest the toxin via the epithelial cells in their midgut by Knowles, B. H. et al. Biochim Biophys Acta (1987) 924:509. Such a mechanism is also supported in part by the results set forth in Example 4 hereinbelow which indicate that the effect of the toxin on embryonic 293 cells modified to express the receptor at their surface is reversible.
Thus, in summary, the invention provides a family of receptors that is able to mediate the negative effects exerted by BT toxin or its modified forms on the cells expressing the receptor, by damaging the cells themselves and/or the tissue or organ of which the cells form a part. The receptor may be expressed natively at the surface of the target cells or the target cells may be modified to contain an expression system which will effect the display of receptor at the surface. The availability of this family of receptors and recombinant methods for its production and for the production of cells displaying it at their surfaces provides a number of opportunities to conduct screening assays for improved toxins, particularly insecticidal toxins, for generation of antibodies that can be useful as alternatives to chemotherapeutic agents for the destruction and/or dissociation of unwanted cells or tissues, and for the design of improved toxins and pharmaceuticals.
Screening Assays
The availability of the recombinant family of receptors of the present invention permits design of straightforward screening assays for toxins which will interact successfully with these receptors resulting in measurable effects on the cells in which the receptors reside. Briefly, suitable host cells, such as COS cells for transient expression, CHO cells for stable expression, and a variety of other mammalian and insect host cells can be modified to contain expression vectors appropriate to the hosts for the production of the receptors of the invention displayed on the surfaces of the cells. Since the receptors are natively membrane proteins, no particular design of the expression system is required in order to effect their disposition at the cell surface. Expression vectors suitable for any desired host are generally known in the art. For example, for mammalian expression, suitable control sequences include the SV40 and adenovirus promoters as constitutive promoters, the metallothionein inducible promoter, suitable enhancers, if desired, and termination signals and the like. For insect cells, the bacculovirus system is preferred. For other eucaryotic cells such as yeast, the glycolytic enzyme promoters and various amino acid synthesis promoters are commonly employed. Procaryotic cells such as E. coli also may be adapted for expression of the receptor in the assay of the invention, for instance by using a reporter gene under the control of cyclic AMP and operably linked to the receptor via protein G such that toxin binding will interrupt adenyl cyclase activity and thereby produce a detectable change in reporter gene activity. The assay system in a prokaryotic host may require further modification to compensate for lack of glycosylation which is known to occur in insect cells where the BT-R1 protein is naturally expressed.
The cells are modified by transfection, retroviral infection, electroporation or other known means, to contain the desired expression system and then cultured under conditions wherein the receptor protein is produced and displayed. If desired, the cells are then recovered from the culture for use in the assay, or the culture itself can be used per se.
In the assays, the modified cells are contacted with the candidate toxin and the effect on metabolism or morphology is noted in the presence and absence of the candidate. The effect may be cytotoxic--i.e., the cells may themselves exhibit one of the indices of cell death, such as reduced thymidine uptake, slower increase in optical density of the culture, reduced exclusion of vital dyes (e.g., trypan blue), increased release of viability markers such as chromium and rubidium, and the like. The differential response between the toxin-treated cells and the cells absent the toxin is then noted. The strength of the toxin can be assessed by noting the strength of the response.
These assays may be conducted directly as described above or competitively with known toxins. For example, one approach might be to measure the diminution in binding of labeled BT cry toxin in the presence and absence of the toxin candidate.
In addition to simply screening candidates, the screen can be used to devise improved forms of toxins which are more specific or less specific to particular classes of insects as desired. The ability to determine binding affinity (Ka and Kd), dissociation and association rates, and cytotoxic effects of a candidate allows quick, accurate and reproducible screening techniques for a large number of toxins and other ligands under identical conditions which was not possible heretofore. Such information will facilitate the selection of the most effective toxins and ligands for any given receptor obtained from any desired host cell.
Competition assays may also employ antibodies that are specifically immunoreactive with the receptor. Such antibodies can be prepared in the conventional manner by administering the purified receptor to a vertebrate animal, monitoring antibody titers and recovering the antisera or the antibody-producing cells for immortalization, to obtain immortalized cells capable of secreting antibodies of the appropriate specificity. Techniques for obtaining immortalized B cells and for screening them for secretion of the desired antibody are now conventional in the art. The resulting monoclonal antibodies may be more effective than the polyclonal antisera as competition reagents; furthermore, the availability of the immortalized cell line secreting the desired antibody assures uniformity of production of the same reagent over time. The information and the structural characteristics of toxins and ligands tested will permit a rational approach to designing more efficient toxins and ligands. Additionally, such assays will lead to a better understanding of the function and the structure/function relationship of both toxin/ligand and BT-R1 analogs. In turn, this will allow the development of highly effective toxins/ligands. Ligands include natural and modified toxins, antibodies (anti-receptor and antiidiotypic antibodies which mimic a portion of a toxin that binds to a receptor, and whatever small molecules bind the receptors.
Therapeutic Strategies
Advantage may be taken of the ability of receptors to mediate the destruction, dissociation or association of cells, tissues or organs by utilizing the screening assay as a method to identify successful therapeutics in the treatment of, for example, malignancies, metastases and infectious microorganisms which naturally express receptors corresponding to BT-R1. The presence of receptors corresponding to the BT-R1 receptor illustrated herein and members of the family of receptors included in the invention in the undesired cells may be exploited by first assessing the interaction of a proposed therapeutic with the receptors on these cells in culture and then identifying agents which successfully interact with the receptors as useful candidate reagents. Antibodies reactive with these receptors comprise a class of promising therapeutic candidates.
In some applications target cells, tissues, organs, and microorganisms which do not express an effective receptor corresponding to the BT-R1 receptor may be transformed or transfected to express an effective corresponding receptor. These targets then will be killed or manipulated with toxin or other ligands. For instance, yeast cells to be used for toxin assays for a particular insect may be transformed with a genetic construct for expression of the receptor from that insect which corresponds to the BT-R1 receptor.
In another aspect of the invention the receptors corresponding to BT-R1 in certain target cells may be manipulated by modified toxin or other ligands to prevent the normal response to toxin (dissociation, damage and death of membranes, cell, tissues and organisms). For instance, a ligand which binds to a corresponding receptor in such a way that normal receptor function is inhibited would thereby prevent the receptor from initiating the usual destructive effects in the presence of a normal ligand such as a toxin. In other words, the invention enables development of competitive inhibitors of a toxin or other ligand which normally initiates destructive or other effects via a receptor corresponding to BT-R1.
The following examples are intended to illustrate but not to limit the invention.
Midguts of M. sexta were extracted and the BT-R1 protein purified according to the method of Vadlamudi, R. K. et al. J Biol Chem (1993) 268:1233, referenced above and incorporated herein by reference. The electroeluted band was confirmed to contain BT-R1 protein by binding to 125 I-cryIA(b) toxin. In gel electrophoresis, the protein bound to toxin had an apparent weight of approximately 210 kD under reducing and nonreducing conditions.
The purified electroeluted BT-R1 was subjected to cyanogen bromide digestion and the. cyanogen bromide fragments separated on a 17% high-resolution tricine SDS-polyacrylamide gel as described by Schagger, H. et al. Anal Biochem (1987) 166:368. The separated fragments were transferred to Problott membranes (Applied Biosystems) and five bands were extracted and subjected to microsequencing using standard instrumentation. The amino acid sequences obtained were:
1. (Met)-Leu-Asp-Tyr-Glu-Val-Pro-Glu-Phe-Gln-Ser-Ile-Thr-Ile-Arg-Val-Val-Ala-Thr-Asp-Asn-Asn-Asp-Thr-Arg-His-Val-Gly-Val-Ala (SEQ ID NO:18);
2. (Met)-X-Glu-Thr-Tyr-Glu-Leu-Ile-Ile-His-Pro-Phe-Asn-Tyr-Tyr-Ala (SEQ ID NO:19);
3. (Met)-X-X-X-His-Gln-Leu-Pro-Leu-Ala-Gln-Asp-Ile-Lys-Asn-His (SEQ ID NO:20);
4. (Met)-Phe/Pro-Asn/Ile-Val-Arg/Tyr-Val-Asp-Ile/Gly (SEQ ID NO:21);
5. (Met)-Asn-Phe-Phe/His-Ser-Val-Asn-Arg/Asp-Glu (SEQ ID NO:23).
An M. sexta cDNA library was constructed from midgut tissue in λgt10 using the Superscript Choice System according to the manufacturer's instructions (Life Technologies, Inc.). Degenerate oligonucleotide probes were constructed based on the peptide sequences determined in Example 1 using the methods and approach described in Zhang, S. et al. Gene (1991) 105:61. Synthetic oligonucleotides corresponding to peptides 1-3 of Example 1 were labeled with γ32 P using polynucleotide kinase and used as probes as described in the standard cloning manual of Maniatis, T. et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2nd ed. 1989). A clone hybridizing to all three probes identified from 40 positive clones as hybridizing to all three of the probes was plaque-purified from a screen of 4�105 recombinants and subcloned into pBluescript (Stratagene). It contained an insert of 5571 bp.
Double-stranded cDNA in pBluescript was sequenced in both directions by the dideoxy termination method with Sequanase (USB) according to the manufacturer's instructions. The sequencing showed an open reading frame of 4584 base pairs or 1528 amino acids along with a polyadenylation signal at position 5561. The sequence obtained and the deduced amino acid sequence is shown in FIG. 1A-1F (SEQ ID NOS: 1 and 2).
Thus, the deduced protein has a molecular mass of 172 kD and a pI of approximately 4.5. The amino acid sequences of the cyanogen bromide fragments of native receptor match perfectly within the deduced amino acid sequence. The open reading frame begins with an ATG that is flanked by the consensus translation initiation sequence GAGATGG for eucaryotic mRNAs as described by Kozak, M. Nucleic Acids Res (1987) 15:8125.
As shown in FIG. 1, the deduced amino acid sequence includes a putative signal, shown underlined, preceding the mature N-terminus Asn-Glu-Arg-etc. Eleven repeats (cad1-cad11) are shown in the extracellular region upstream of the membrane domain, shown with the heavy underline, at positions 1406-1427. The end of the 11th repeat is shown with an arrowhead. The positions of the five CNBR fragments are also shown under the complete sequence.
Like known cadherins, the external domain of BT-R1 is highly repetitive and contains 11 repeats (cad1-cad11; see FIGS. 2A-2F (SEQ ID NO:2)). The greatest similarity of BT-R1 to the cadherins is with the extracellular repeats of the cadherin motif of mouse P-cadherin, Drosophila fat tumor suppressor and the protocadherins, although homologies are not high (20-40 homology and 30-60 percent similarity). The conserved repeats of BT-R1 included AXDXD SEQ ID NO:23), DXE, DXNDXXP (SEQ ID NO:24), one glutamic acid residue and two glycine residues. Motifs A/VXDXD (SEQ ID NO:25), DXNDN (SEQ ID NO:26) are the consensus sequences for calcium binding and two such regions are present in a typical cadherin repeat. In all repeats of BT-R1, the sequence DXNDN (SEQ ID NO:26) is preceded by 8 to 14 hydrophobic amino acids. Similar hydrophobic sequences also have been observed in the cadherins. The length of the hydrophobic stretches suggests that these areas are not transmembrane regions but that they represent β-sheet structures commonly present in cadherin-like repeats. BT-R1 contains a putative cytoplasmic domain of 101 amino acids, smaller than vertebrate cadherin cytoplasmic domains (160 amino acids), and shows no homology to any of the cadherin cytoplasmic domains or to cytoplasmic domains of other proteins to which it has been compared in a current sequence data base.
To confirm that the sequenced clone encoded full-length BT-R1 protein, total mRNA was prepared from midguts of M. sexta subjected to Northern blot by hybridization with the antisense 4.8 kb SacI fragment of the BT-R1 cDNA clone. The Northern blot analysis was conducted by hybridizing to the antisense probe at 42� C. and 50% formamide, 5� Denhardt's Reagent, 5� SSCP and 50 μg/ml salmon sperm DNA. The filter was then washed two times with 1� SSC+0.1% SDS and two times with 0.15� SSC+0.1% SDS at 42� C. Each wash was roughly 20 minutes. The filter was then exposed to X-ray film for 24 hours. The 4.8 kb probe hybridized to a single 5.6 kb band.
The BT-R1 clone was translated using rabbit reticulolysate and the resulting translated products were immunoprecipitated with antisera raised against native protein encoded by BT-R1. For the in vitro translation, pBluescript plasmid containing BT-R1 cDNA was linearized and transcribed with T3 polymerase (Pharmacia). The translation was conducted according to manufacturer's instructions with nuclease-treated rabbit reticulolysate (Life Technologies, Inc.). After one hour of incubation at 30� C., the reaction mixture was combined with an equal volume of SDS buffer or lysed with 50 mM Tris buffer containing 1% NP40 and 250 mM NaCl (pH 8.0) for immunoprecipitation. Preimmune serum was used as a control. Translation and immunoprecipitation products were electrophoresed on a 7.5% SDS-polyacrylamide gel fixed, treated with Enhance (Dupont NEN), dried and exposed to X-ray film for 12 hours.
Two protein bands of approximately 172 kD and 150 kD as determined by SDS-PAGE were obtained; it is postulated that the 150 kD translation product was due to initiation of translation from an internal methionine at amino acid 242. This is consistent with the observations of Kozak, M. Mol Cell Biol (1989) 9:5073.
Thus, both results confirm that a full-length clone was obtained.
The BT-R1 cDNA clone was subcloned into the mammalian expression vector pcDNA3 (Invitrogen) and the construct transfected into COS-7 cells. Membranes isolated from the COS-7 transfectants were solubilized, electrophoresed and ligand blotted with 125 I-cryIA(b) toxin. The cells were harvested 60 hours after transfection, washed with phosphate-buffered saline and lysed by freezing in liquid nitrogen. Cell membranes were prepared by differential centrifugation as described by Elshourbagy, N. A. et al. J Biol Chem (1993) 266:3873. Control cells were COS-7 cells transfected with pcDNA3.
The cell membranes (10 μg) were separated on 7.5% SDS-PAGE blotted to a nylon membrane and blocked with Tris-buffered saline containing 5% nonfat dry milk powder, 5% glycerol and 1% Tween-20. The nylon membrane was then incubated with 125 I-cryIA(b) toxin (2�105 cpm/ml) for two hours with blocking buffer, dried and exposed to X-ray film at -70� C. The labeled toxin bound to a 210�5 kD protein; the 210 kD band was observed only in lanes containing membranes prepared from either M. sexta or COS-7 cells transfected with the BT-R1 cDNA construct containing 4810 bp of cDNA comprising the open reading frame.
The discrepancy between the 210 kD protein expressed and the calculated 172 kD molecular weight is due to glycosylation of the protein; in vitro translation of the cDNA clone, as described above, which does not result in glycosylation, does produce the 172 kD protein. To verify this, the COS-7 produced protein was subjected to digestion with N-glycosidase-F by first denaturing the purified protein by boiling in 1% SDS for 5 minutes followed by addition of NP-40 to a final concentration of 1% in the presence of 0.1% SDS, and then incubating the denatured protein in sodium phosphate buffer, pH 8.5 at 37� C. with N-glycosidase-F for 10 hours. Controls were incubated under the same conditions without enzyme. Digestion products were separated on a 7.5% SDS-PAGE and stained with Coomassie brilliant blue. This glycosidase treatment reduced the molecular weight of BT-R1 protein from 210 to 190 kD; this indicates N-glycosylation at some of the 16 consensus N-glycosylation sites in the protein. Treatment of BT-R1 with O-glycosidase and neuraminidase did not alter the mobility of the protein.
In addition, embryonic 293 cells were transfected with the BT-R1 cDNA clone in pcDNA3 and incubated with the labeled toxin (0.32 nM) in the presence of increasing concentrations (0 to 106 M) of unlabeled toxin. Nonspecific binding was measured as bound radioactivity in the presence of 1 μM unlabeled toxin. A value for the dissociation constant (Kd) of 1015 pM was determined by Scatchard analysis; this is approximately the same value that was obtained for the natural receptor as described by Vadlamudi, R. K. et al. J Biol Chem (1993) (supra).
Both unmodified embryonic 293 cells, and 293 cells which have been modified to produce the BT-R1 receptor as described in Example 3, when cultured in vitro form adherent star-shaped clusters. When BT toxin (200 nM) is added to serum-free medium, tee clusters round up and release from the plastic surfaces of the culture dish. This effect is also observed under known conditions of cytotoxicity for 293 cells. The foregoing effect is observed only when the cells are cultured in serum-free medium since the toxin binds to serum and would thus be ineffective under conditions where serum is present.
However, in the presence of anti-receptor antisera, this effect of BT toxin is blocked. Also, when serum is added back to a culture of modified E293 cells which has been treated in serum-free conditions with the toxin, the cells revert to their normal star-shaped adherent cluster shapes. This indicates that the effect of the toxin is reversible.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 26(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5577 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 197..4780(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:[encoded data, 11594 characters](2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1528 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:[encoded data, 5586 characters](2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 107 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GluTrpValMetProProIlePheValProGluAsnGlyLysGlyPro151015PheProGlnArgLeuAsnGlnLeuLysSerAsnLysAspArgGlyThr202530LysIlePheTyrTyrSerIleThrGlyProGlyAlaAspSerProPro354045GluGlyValPheThrIleGluLysGluSerGlyTrpLeuLeuLeuHis505560MetProLeuAspArgGluLysIleValLysTyrGluLeuTyrGlyHis65707580AlaValSerGluAsnGlyAlaSerValGluGluProMetAsnIleSer859095IleIleValThrAspGlnAsnAspAsnLysPro100105(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 105 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GluAspThrValTyrSerPheAspIleAspGluAsnAlaGlnArgGly151015TyrGlnValGlyGlnIleValAlaArgAspAlaAspLeuGlyGlnAsn202530AlaGlnLeuSerTyrGlyValValSerAspTrpAlaAsnAspValPhe354045SerLeuAsnProGlnThrGlyMetLeuThrLeuThrAlaArgLeuAsp505560TyrGluGluValGlnHisTyrIleLeuIleValGlnAlaGlnAspAsn65707580GlyGlnProSerLeuSerThrThrIleThrValTyrCysAsnValLeu859095AspLeuAsnAspAsnAlaProIlePhe100105(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 93 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:AlaSerProValIleThrLeuAlaIleProGluAsnThrAsnIleGly151015SerLeuPheProIleProLeuAlaSerAspArgAspAlaAsnGluLeu202530GlnValAlaGluAspGlnGluGluLysGlnProGlnLeuIleValMet354045GlyAsnLeuAspArgGluArgTrpAspSerTyrAspLeuThrIleLys505560ValGlnAspGlyGlySerProProArgAlaThrSerAlaLeuLeuArg65707580ValThrValLeuAspThrAsnAspAsnAlaProLysPhe8590(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 106 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:IleValThrGluAsnIleTrpLysAlaProLysProValGluMetVal151015GluAsnSerThrProHisProIleLysIleThrGlnValArgTrpAsn202530AspProGlyAlaGlnTyrSerLeuValAspLysGluLysLeuProArg354045PheProPheSerIleAspGlnGluGlyAspIleTyrValThrProLeu505560AspArgGluGluLysAspAlaTyrValPheTyrAlaValAlaLysAsp65707580GluTyrGlyLysProLeuSerTyrProLeuGluIleHisValLysVal859095LysAspIleAsnAspAsnProProThrCys100105(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 105 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:IleThrAlaAsnLeuGlyThrGlnValIleTyrMetAspGluGluIle151015GluAspGluIleThrIleAlaIleLeuAsnTyrAsnGlyProSerThr202530ProPheIleGluLeuProPheLeuSerGlySerTyrAsnLeuLeuMet354045ProValIleArgArgValAspAsnGlySerAlaSerHisHisHisAla505560ArgGlnHisTyrGluLeuProGlyMetGlnGlnTyrMetPheAsnVal65707580ArgValAspGlyGlnSerLeuValAlaGlyValSerLeuAlaIleVal859095AsnIleAspAspAsnAlaProIleIle100105(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 113 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GlnAsnPheGluProCysArgValProGluLeuGlyGluProGlyLeu151015ThrGluCysThrTyrGlnValSerAspAlaAspGlyArgIleSerThr202530GluPheMetThrPheArgIleAspSerValArgGlyAspGluGluThr354045PheTyrIleGluArgThrAsnIleProAsnGlnTrpMetTrpLeuAsn505560MetThrIleGlyValAsnThrSerLeuAsnPheValThrSerProLeu65707580HisIlePheSerValThrAlaLeuAspSerLeuProAsnThrHisThr859095ValThrMetMetValGlnValAlaAsnValAsnSerArgProProArg100105110Trp(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 106 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:LeuGluIlePheAlaValGlnGlnPheGluGluLysSerTyrGlnAsn151015PheThrValArgAlaIleAspGlyAspThrGluIleAsnMetProIle202530AsnTyrArgLeuIleThrAsnGluGluAspThrPhePheSerIleGlu354045AlaLeuProGlyGlyLysSerGlyAlaValPheLeuValIleAspArg505560AspThrLeuGlnArgGluValPheProLeuThrIleValAlaTyrLys65707580TyrAspGluGluAlaPheSerThrSerThrAsnValValIleIleVal859095ThrAspIleAsnAspGlnArgProGluPro100105(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 119 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:IleHisLysGluTyrArgLeuAlaIleMetGluGluThrProLeuThr151015LeuAsnPheAspLysGluPheGlyPheHisAspLysAspLeuGlyGln202530AsnAlaGlnTyrThrValArgLeuGluSerValAspProProGlyAla354045AlaGluAlaPheTyrIleAlaProGluValGlyTyrGlnArgGlnThr505560PheIleMetGlyThrLeuAsnHisSerMetLeuAspTyrGluValPro65707580GluPheGlnSerIleThrIleArgValValAlaThrAspAsnAsnAsp859095ThrArgHisValGlyValAlaLeuValHisIleAspLeuIleAsnTrp100105110AsnAspGluGlnProIlePhe115(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 104 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GluHisAlaValGlnThrValThrPheAspGluThrGluGlyGluGly151015PhePheValAlaLysAlaValAlaHisAspArgAspIleGlyAspVal202530ValGluHisThrLeuLeuGlyAsnAlaValAsnPheLeuThrIleAsp354045LysLeuThrGlyAspIleArgValSerAlaAsnAspSerPheTyrHis505560ArgGluSerGluLeuPheValGlnValArgAlaThrAspThrLeuGly65707580GlnProPheHisThrAlaThrSerGlnLeuValIleArgLeuAsnAsp859095IleAsnAsnThrProProThrLeu100(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 138 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:ArgLeuProArgGlySerProGlnValGluGluAsnValProAspAla151015HisValIleThrGlnGluLeuArgAlaThrAspProAspThrThrAla202530AspLeuArgPheGluIleAsnTrpAspThrSerPheAlaThrLysGln354045GlyArgGlnAlaAsnProAspGluPheArgAsnCysValGluIleGlu505560ThrIlePhePheProGluIleAsnAsnIleAsnAsnArgGlyLeuAla65707580IleGlyArgValValAlaArgGluIleArgHisAsnThrIleAspTyr859095GluGluPheGluValLeuSerLeuThrValArgValArgAspLeuAsn100105110ThrValTyrGlyAspAspTyrAspGluSerMetLeuThrIleThrIle115120125IleAspMetAsnAspAsnAlaProValTrp130135(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 124 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:ValGluGlyThrLeuGluGlnAsnPheArgValArgGluMetSerAla151015GlyGlyLeuValValGlySerValArgAlaAspAspIleAspGlyPro202530LeuTyrAsnGlnValArgTyrThrIlePheProArgGluAspThrAsp354045LysAspLeuIleMetIleGluLeuProHisGlySerAsnPheArgGlu505560HisLysArgArgIleAspAlaAsnThrProProArgPheHisLeuTyr65707580TyrThrValValAlaSerAspArgCysSerThrGluAspProAlaAsp859095CysProProAspProTyrTyrTrpGluThrGluGlyAsnIleThrIle100105110HisIleThrAspThrAsnAsnLysValProGlnAla115120(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 122 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GluThrThrLysPheAspThrValValTyrIleTyrGluAsnAlaThr151015HisLeuAspGluValValThrLeuIleAlaSerAspLeuAspArgAsp202530GluIleTyrHisMetValSerTyrValIleAsnTyrAlaValAsnPro354045ArgLeuMetAsnPhePheSerValAsnArgGluThrGlyLeuValTyr505560ValAspTyrGluThrGlnGlySerGlyLeuAspArgAspGlyAspGlu65707580ProThrHisArgIlePhePheAsnLeuIleAspAsnPheMetGlyGlu859095GlyGluGlyAsnArgAsnGlnAsnAspThrGluValLeuValIleLeu100105110LeuAspValAsnAspAsnAlaProGluLeu115120(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 146 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:ProProProSerGluLeuSerTrpThrIleSerGluAsnLeuLysGln151015GlyValArgLeuGluProHisIlePheAlaProAspArgAspGluPro202530AspThrAspAsnSerArgValGlyTyrGluIleLeuAsnLeuSerThr354045GluArgAspIleGluValProGluLeuPheValMetIleGlnIleIle505560AlaAsnValThrGlyTyrGluIleLeuAsnLeuSerThrGluArgAsp65707580IleGluValProGluLeuPheValMetIleGlnIleAlaAsnValThr859095GlyGluLeuGluThrAlaMetAspLeuLysGlyTyrTrpGlyThrTyr100105110AlaIleTyrIleLeuAlaPheAspHisGlyIleProGlnMetSerMet115120125AsnGluThrTyrGluLeuIleIleHisProPheAsnTyrTyrAlaPro130135140GluPhe145(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 120 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:ValPheProThrAsnAspAlaValIleArgLeuAlaArgGluArgAla151015ValIleAsnGlyValLeuAlaThrValAsnGlyGluPheLeuGluArg202530IleSerAlaThrAspProAspGlyLeuHisAlaGlyValValThrPhe354045GlnValGlyAspGluGluSerGlnArgTyrPheGlnValValAspAsn505560AspGlyGluAsnLeuGlySerLeuArgLeuLeuGlnAlaValProGlu65707580GluIleArgGluPheArgIleThrIleArgAlaThrAspGlnGlyThr859095AspProGlyProLeuSerThrAspMetThrPheArgValValPheVal100105110ProThrGlnGlyGluProArgPhe115120(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 112 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:AlaSerSerGluHisAlaValAlaPheIleGluLysSerAlaGlyMet151015GluGluSerHisGlnLeuProLeuAlaGlnAspIleLysAsnHisLeu202530CysGluAspAspCysHisSerIleTyrTyrArgIleIleAspGlyAsn354045SerGluGlyHisPheGlyLeuAspProValArgAsnArgLeuPheLeu505560LysLysGluLeuIleArgGluGlnSerAlaSerHisThrLeuGlnVal65707580AlaAlaSerAsnSerProAspGlyGlyIleProLeuProAlaSerIle859095LeuThrValThrValThrValArgGluAlaAspProArgProValPhe100105110(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:MetLeuAspTyrGluValProGluPheGlnSerIleThrIleArgVal151015ValAlaThrAspAsnAsnAspThrArgHisValGlyValAla202530(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:MetXaaGluThrTyrGluLeuIleIleHisProPheAsnTyrTyrAla151015(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:MetXaaXaaXaaHisGlnLeuProLeuAlaGlnAspIleLysAsnHis151015(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2(D) OTHER INFORMATION: /note= "This position is Phe/Pro"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 3(D) OTHER INFORMATION: /note= "This position is Asn/Ile"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 5(D) OTHER INFORMATION: /note= "This position is Arg/Tyr"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 8(D) OTHER INFORMATION: /note= "This position is Ile/Gly"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:MetXaaXaaValXaaValAspXaa15(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 4(D) OTHER INFORMATION: /note= "This position is Phe/His"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 8(D) OTHER INFORMATION: /note= "This position is Arg/Asp"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:MetAsnPheXaaSerValAsnXaaGlu15(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:AlaXaaAspXaaAsp15(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:AspXaaAsnAspXaaXaaPro15(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 1(D) OTHER INFORMATION: /note= "Alanine(A) or Valine(V)"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:XaaXaaAspXaaAsp15(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:AspXaaAsnAspAsn15__________________________________________________________________________
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