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14 Toilet Paper Roll Craft Ideas
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Star Paper Topiary
By: Diane Flowers for STYROFOAM Brand Foam
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It's so easy to create this Star paper topiary. Recycle gift wrap from the holidays or birthdays or use some of your favorite scrapbook paper to make this paper crafted decoration. Makes a perfect centerpiece!
- STYROFOAM Brand Foam:
- Star, 12", 9" or 6", one
- Vase Insert to fit pot, one
- Holiday gift wrap
- Paper mâché pot or pot of your choice, 6" for large or medium topiary, 4" for small
- Wooden dowel, 1/4"
- One bag of each color of Reindeer moss: green; red
- Paint pen, Metallic gold
- Serrated knife
- Paraffin or candle stub
- Pencil with eraser
- Measuring tape
- Scissors
- Wrinkle-free paper glue
- Low temperature glue gun and glue sticks
- Glue foam vase insert into pot. Wax serrated knife with candle stub or paraffin; cut vase insert level with top of pot.
- Trace foam star twice onto wrong side of desired paper, noting position of motifs to be sure design is centered. Cut out paper stars.
- Apply small dots of paper glue to backs of paper stars and glue to front and back of foam star.
- Mark opposite sides of pot rim with pencil. Measure and cut paper to cover each half of pot, below rim, again noting motif position.
- Center motifs over pencil marks drawn on rim and glue to pot using paper glue. Cut and glue small pieces of paper to pot to fill any gaps.
- Measure pot rim and cut strip of paper to cover it. Glue paper to rim.
- Use paint pen to highlight star and pot, including rim, as desired; let dry.
- Use glue gun to glue small pieces of green reindeer moss to star edges and lower edge of pot rims. Glue small pieces of red moss into green, every 2", around star. Use scissors to trim moss as needed.
- Cut dowel as follows: 14" length for large topiary; 12" for medium; 9" for small. Glue and insert dowel into center of vase insert in pot; let dry.
- Push star onto dowel so designs on star align with designs on pot. Remove stars, apply glue to top of dowel and replace star; let dry.
- Glue green moss to exposed areas of vase insert; let dry.
®™ Trademark of The Dow Chemical Company ("Dow") or an affiliated company of Dow.
For more project ideas, please see styrofoamstyle.com.
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Harrell Leads (4) Louisville Past (8) NC State
Clippers trounce lowly Sixers, 119-98
PHILADELPHIA -- It was a locker room devoid of champagne, commemorative T-shirts or even congratulatory chatter, the Los Angeles Clippers acting as if they had done nothing more than log a late-season victory over one of the worst teams in the NBA. The significance of their 119-98 victory over the Philadelphia 76ers on Friday night at Wells Fargo Center seemed universally lost on their star players. "We clinched tonight?" point guard Chris Paul asked reporters inquiring about the Clippers having secured a playoff berth. "I didn't know that." Going to the playoffs is nothing new for these Clippers. They have qualified for the postseason for a fourth consecutive year for the first time ...

subset: commoncrawl


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When things are at their bleakest is when it gets super bleak then light comes then total darkness, a little light, then death.
Feb 11, 2014 @ 1:58pm
These Winter Olympics need to mix it up. I want to see rival curling teams get into a broom fight.


text: Mar 25, 2014 13:04 18-wheeler removed; I-110 S. ramp to I-10 W. reopens 18-wheeler removed; I-110 S. ramp to I-10 W. reopens Advocate staff report March 25, 2014 Comments The ramp from Interstate 110 South to Interstate 10 West has reopened after being blocked for several hours Monday, because of an overturned 18-wheeler, the Baton Rouge Police Department announced. Cpl. Don Coppola announced before 1:45 p.m. that the ramp had finally cleared. The truck driver was cited for unsecured load, Coppola said. "It appears something shifted in his load which caused him to turn over." No one was injured, Coppola said. Get The Advocate in your inbox Subscribe


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Pre-Ceremony -
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|Mar 12, 2012, 06:10 PM
I realize it's disheartening when it's not as you expected. But once you get her up and going you'll be so glad you did. Last I heard instructions are being written but aren't completed yet. Email Fred and he'll send you what he has I'm sure.
You beat me to asking for help but I was going to give you my cell number and ask you to call anytime, none of us never know when I'm available, so just call and I'll answer if I am. Work has been slow lately! I'll send you an email with my phone number. Since you have my email we can exchange photos with questions you have and that will help me explain what to do. It'll be easier than you think.
I know you don't want to hear this, but It sounds quite typical of a garage built glider. Actually, it sounds about exactly what the purchaser of my ALES eSupra will have to do.
The motor mount is easy. I have tips for that. The hinges are very easy, it's called tape. There is s specific kind I recommend. The servos in the wing should be easy, but I may need a photo from you. The servos for the rudder/elevator do they go in the tail or up in the nose behind the motor? Either way that's easy to.
We'll just break this down into one step at a time and next thing you know you'll be flying, sooner than you think too!
|Mar 12, 2012, 06:10 PM
I am sorry to hear you are not as pleased with plane as hoped. When and if you come over to visit Curtis, I can come by also and lend a hand with the finishing steps. We can tag team it and probably get it put together pretty quick. If Curtis is up for it. A little glue and large shake-n-bake bag should get most of it taken care of in a hurry, LOL!
I am sure when it is done you will be quite pleased with it.
|Mar 12, 2012, 06:13 PM
I'd like more detail on how you did the graphics, please...?
Curtis "In Awe" Suter
|Mar 13, 2012, 03:41 PM
Guys (and Gals?),
Just a very short update on my currently delayed project. A couple of my Helena friends have been extremely kind in offering to get my Oracle project off the ground, and I may just take them up on their offer. Thank you so much!
Anyhow, I've attached a few pictures of what I'm facing with this project. I know that I've built and assembled things more difficult than this, but as I told Curtis, my biggest frustration is having no space or workbench to attack this thing, as well as zero instructions sent with this ARF. Hope you find these pix interesting at the very least. Enjoy!
|Mar 13, 2012, 04:13 PM
United States, MT, Belgrade
Joined Aug 2010
344 Posts
|Mar 13, 2012, 06:00 PM
From what I can tell, the horizontal mounts on top of the boom (above the slot) with the control arm extending down into the boom attaching to the pushrod. But really not sure how the rudder pushrod will mount, since the pushrod comes out the side and the rudder is above the horizontal. Other than that, it looks like a pretty straight forward build. Oh and that roll of tape is the hinge tape for your surfaces.
I can completely relate to limited work space, a lot of times I use the dining room table, but that is not the best situation. Can lead to clutter and less than excited better half, HAHA! I do think we should have build day here in Helena. If someone can volunteer some workspace I need to get going on my project also. Need some gear for it though.
Well, I hope all is
|Mar 13, 2012, 06:02 PM
It sure would make time away from the family go faster if you could find a place to fly. I hope the job is all it's cracked up to be and you are making big bucks.
Post some pictures if you can,
Take care and best of luck to you.
|Mar 15, 2012, 09:16 PM
United States, MT, Belgrade
Joined Aug 2010
344 Posts
|Mar 16, 2012, 01:48 PM
Nope - in fact, as I type, it is still sitting on the floor behind the couch where you last saw it There are, however, some plans being made by our friends over in our state capital to get it airborne sooner than I personally planned. Sooooo, you can just sit back and watch it come to fruition
(I'm sure that wasn't me yelling at the refs in Ashville. Although I'm trying to keep in touch with most of the games as they unfold, I'm also trying to keep my mouth shut and not holler at the tv during the games.)
Excited about my 'Alice' plane!!!
|Mar 17, 2012, 08:13 AM
|Mar 19, 2012, 12:36 PM
Sorry and Happy
To all my Helena Slope Soaring and ALES friends,
First of all, I want to apologize for not making it over today. I truly feel bad about having all of you set the whole day aside to help me with my Oracle project, and then not make it over. Truth be known, I was on my way down the interstate toward Helena @7:30 this morning, in the middle of that horrible snow storm, when a phone call caused me to turn around and head back to Bozeman. My daughter, who was not due until next week, gave birth to her first child, (and our new granddaughter), at 9:11 am this morning. Of course I needed to be there............so, I think I have a good excuse - at least this time around
Anyhow, if you are still willing, my schedule is sorta flexible, and I'd like to re-schedule when Curtis can be there. I much appreciate all of your expertise in those areas that I don't have a clue. Let me know if that is doable. Thank you!
|Mar 19, 2012, 12:55 PM
Well Congratulations to your daughter and her new family and to you and your wife as well!!!! That is awesome news! May I assume that baby and mom are both doing well
Absolutely no apologies necessary, I am surprised you were willing to attempt the drive here this morning. Roads were less than desirable here in East Helena for sure.
I am free to help out whenever you are free to come over Arlen, just let me know and I will be there.
For now have a joyful Monday!
|Mar 19, 2012, 10:48 PM
Tis that time of year again
My daughter Mikalae has finished her bridge project for school. Her sister did this last year and her bridge held 57lbs of pressure before it exploded. So we are hoping of course to best that weight. Fun working with balsa and kids
I hope all is well with everyone,
|Mar 20, 2012, 01:04 PM
Guys (and Gals),
Attached is a picture of why I couldn't make it over to Helena on Monday. It was well worth the detour.
|Mar 20, 2012, 01:49 PM
United States, MT, Belgrade
Joined Aug 2010
344 Posts
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|Great Falls, Va 7:30am - March 30th
|Aerial Photography
|Jan 05, 2015 09:35 AM


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RECIPE: love chicken and artichokes.......do you have some reci
Chicken Artichoke Jack Casserole
10 chicken breasts, deboned and skinned
10 strips Monterrey Jack cheese
1 egg, beaten
1 package Pepperidge Farm Dressing Mix
3/4 to 1 cup chopped parsley
1 teaspoon garlic salt
1 can mushroom soup
1 can cream of celery soup
1 can cream of chicken soup
6 ounces dry white wine
1/2 pound fresh mushrooms, sliced
1/2 to 3/4 cup Parmesan cheese, shredded
Preheat oven to 300F. Pound chicken breasts flat between two sheets of waxed paper. Roll each breast around a large strip of Jack cheese. Dip each in beaten egg. Place in large glass baking dish that has been sprayed with non- stick coating. Sprinkle with dressing mix, parsley and garlic salt. Combine soups, wine and mushrooms. Spread over chicken. Place artichoke hearts that have been sliced in half over the top. Sprinkle with enough Parmesan cheese to cover. Bake for 1-1/2 hours.
Serves 6-8.
This favorite recipe is for a clay pot or Romertopf, but you could make it in any covered casserole and bake at a lower temperature.
Baked Chicken and Artichokes
1/4 lb. small mushrooms
1 small onion, chopped fine
1 clove garlic, minced
3 lbs. chicken pieces, skin off
2 TB flour
1 1/2 t. salt
1/2 t. paprika
1/4 t. chopped rosemary
dash white pepper
1/2 cup chicken broth
1/4 cup sherry
1 can artichoke hearts, drained
If using clay pot, soak top and bottom in sink of water for 15 minutes. Drain.
If using covered casserole, lightly grease or spray with Pam.
Put mushrooms, onion, garlic in pot or casserole. Coat chicken pieces with a mixture of flour, salt, paprika, rosemary, pepper. Put chicken on top of vegetables. Pour in chicken broth and sherry.
Put lid on pot/casserole. If using clay pot, put in a cold oven and turn heat on to 450F. If using casserole, put in a preheated 375F oven. Bake 1 hour for either method.
Stir in artichokes gently after baking 1 hour. Bake 8-10 minutes more covered. Remove cover and bake 5-10 minutes more until chicken starts to crisp.
Serves 6
We like this one - you can substitute sherry for the wine if you like.
Lompoc, CA
* Exported from MasterCook *
Company Chicken-Artichoke Bake
Recipe By :
Serving Size : 6 Preparation Time :0:00
Categories :
Amount Measure Ingredient -- Preparation Method
-------- ------------ --------------------------------
1/4 cup butter
6 skinned and boned chicken breast halves
1 cup chopped onion
1 clove minced garlic
3 tablespoons flour
1/4 teaspoon salt
1 1/4 cups chablis
1 cup chicken broth
3 ounces cream cheese
14 ounces artichoke hearts -- drained
1 pound sliced mushroom
Melt butter in a large skillet over medium heat. Add chicken, and cook 5 minutes on each side or until browned. Remove chicken from skillet, reserving pan drippings; place chicken in a 2-quart casserole. Set aside.
Saute onion and garlic in reserved pan drippings until tender. Add flour and salt, stirring until smooth. Cook 1 minute, stirring constantly. Gradually add wine and broth; cook over medium heat, stirring constantly, until mixture is thickened and bubbly. Add cream cheese, stirring until cheese melts. Remove from heat; set aside.
Arrange halved artichoke hearts over chicken. Spoon cream cheese mixture over chicken and artichokes. Cool.
TO STORE: Cover and refrigerate up to 24 hours.
TO SERVE: Bake, covered, at 350F for 45 to 50 minutes or until bubbly. Sprinkle with toasted almonds.
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Middle School Is Worse Than Meatloaf
A Year Told Through Stuff
From the critics
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I loved it, read it in less than 4 days,
so fun and easy to read
i love it!
My daughter could not put this down .... I wish there were more books in this series.
This was a really entertaining read, a very creatie way to represent a story. Though I wish the story could've ended in a more ''complete'' way. Because I dont know what happened to some people in the end.
Cute book, talks about a girls seventh grade expeience.
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USGS: More Data Needed To Assess Any Fracking-Pollution Link
A new study by the U.S. Geological Survey says more data is needed to be able to say for sure if a link exists between unconventional oil and gas development and degraded water quality.
Keystone XL Veto Could Make A Case Against Fracking
The move to veto showcases the Obama administration's focus on climate change and sheds light on the growing concerns over fracking.
Defying GOP, Obama Vetoes Keystone XL Pipeline Bill
Defying the Republican-run Congress, President Barack Obama rejected a bill Tuesday to approve construction of the Keystone XL oil pipeline, wielding his veto power for only the third time in his presidency.
Congressional Democrats Seek To Step Up Fracking Oversight
Democrats on a congressional oversight panel are stepping up their investigation into how well states are regulating the disposal of oil and gas waste, citing continuing public concern about the potential environmental and health risks of hydraulic fracturing.
Drinking Dirty Water: Is America's Water Safety In Jeopardy?
Despite the Safe Drinking Water Act, you and your family may be drinking dirty water.


text:
(SPOILERS, SPOILERS!) Turning at every corner. Captain America: The Winter Soldier tie-in episode! (SPOILERS, SPOILERS!)
With the rise of Hydra in Captain America: The Winter Soldier, the Agents of S.H.I.E.L.D. are facing off against a large scale threat.
This tie-in episode wasn't entirely bad. With the Thor: Dark World tie-in episode a couple of episodes ago, you can tell that the creative minds behind this show have stepped up their game with this episode. However, it's still in question of "how far have they stepped up their game?" The answer is simple: some.
In this episode, everything has gone from bad-to-worse for Coulson and his Agents of S.H.I.E.L.D. in almost an instant. From the eyes of Coulson it looks like his partner, Melinda May, is actually a double agent and has been spying on the entire team since it's establishment. However, all is not as it seems. With the end of Captain America: The Winter Soldier, Hydra has been working for nearly 70 years to infiltrate S.H.I.E.L.D. and corrupt it from within; this plan has worked! With the apparent demise of Nick Fury, S.H.I.E.L.D. is without it's one good eye and moral compass; chaos insures with Hydra raining fire on all that oppose it! -- and Coulson and company are right in the middle of it.
In my mind, this is probably the best episode so far. The title of this episode "Turn, Turn, Turn" is appropriate, as a lot of story elements (i.e. the Clairvoyant, Hydra, etc.) have you changing your mind a lot -- in a good way. The action has finally gotten better (the death count in this series is still extremely low as for some reason the Agents refuse to kill), but moving past that we get to see drawn out fisticuffs and some good standoffs/shootouts.
"The Clairvoyant" is a name/villain that has always been one step ahead of Coulson's Agents of S.H.I.E.L.D. He, or she, is the reason we've had Deathlok and a bunch of other weird things happen that the Agents of S.H.I.E.L.D. have had to clean up. The Clairvoyant has also been greatly intrigued by the apparent return of Coulson, however didn't know how it exactly happened. Before I get into all of that, It does connect with Hydra and the Marvel Phase (movie) project; so calm down.
I will say that this episode having piggybacked on the ending of Captain America: The Winter Soldier, it did help the series a little. Everything that's happened so far with trust issues between the team members, Coulson's resurrection explained, and the Clairvoyant being this threat that's always been out of reach; it never really had a strong footing and it felt a bit like each episode was self contained (only hoping that it would workout in the end). NOW, with Cap 2 having been released everything that's happened so far makes sense.
The writers had been spinning a web (no relation to Sony's Spider-Man) that made us question if Coulson was the Clairvoyant or someone else was. It could've easily turned out to be Coulson as the Clairvoyant, because he was revived inhumanely and could've had a split personality. While that would've been cool, I still like Coulson on the side of justice. Eventually it turned out, from this episode, that Coulson wasn't the Clairvoyant and neither was Victoria Hand nor Melinda May. Instead it was Agent Garret (double agent for Hydra) who turned out to be the Clairvoyant; after the team found out that it was a S.H.I.E.L.D. agent leaking information.
The Clairvoyant wanting to know how Coulson came back to life was (somewhat) key in a part of Hydra's plan. Coulson was revived thanks to an alien specimen known as; Kree -- this alien's physiology brought Coulson from the grave. So obviously it would be helpful to Hydra's longterm plan to have a serum that could bring back it's soldiers better than ever. While that reveal was underwhelming for the series as a whole, the experience of finding out was quite exciting. For once, it felt like the Agents of S.H.I.E.L.D. were relevant in the Marvel (move phase); even though their part is like a corner in the larger universe.
Overall it was a good episode. It had it's ups-and-downs and just this once it felt like the Agents of S.H.I.E.L.D. were a real part of the Marvel universe, and hopefully the writers can continue this momentum (for the series sake). There was a nice twist at the end that had Agent Ward secretly be an agent of Hydra as well. So that is one thing to look forWARD to (see what I did there?) Anyway, the acting was good, the story was surprisingly better than the last couple of episodes (T.R.A.C.K.S. is still one of the best episodes), and we as Marvel watchers have something to look forWARD to. 5 out of 5.
Until the next episode!
Last review from this series (and other reviews):
Agents of S.H.I.E.L.D. "End of the beginning" REVIEW
Last review --- Wonder Woman #29
Last review ---- X-Men (volume 4, 2014) #12
You can complain, or compliment, me on Twitter (@Lol_Clark) or here on Comic Vine (@Super-Man 23). All of my daily reviews are here: -click me-


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Room Divider Ideas
Have a four-panel wooden room divider which is empty. Want a light and cheerful look on both sides. Have wondered about fabric, of course, and even wall paper. Suggestions for something different would be greatly appreciated.
mirror tiles...decoupage...hang pictures on it or attach them to the wood.
I have two room dividers which currently have anaglypta type wallpaper that I did a paint treatment on. However recently in a store I saw a divider made of 3 vintage doors that had gathered sheer fabric. I'm planning to do that with one of mine - it was a lovely soft look for a bedroom. I'm planning to glue old dress patterns in each section of the other one.
Other ideas:
~Stretch yarn or twine between tacks from top to bottom. Or buy one of those bead curtains and use the beaded strings.
~For a rustic look attach twigs.
~Fretwork panels are available at home improvement stores.
~Bamboo roll-up blinds could be cut and attached.
~Attach heavy rolled decorative gift wrap.
One can often find ideas for projects by wondering around building supply, craft, or dollar stores. Or let your fingers wonder the internet! :)


text:
I stayed here for one night en route to Malaga Airport. The twin room was large and comfy. I think most rooms have a view but our room was on the first floor so didn't see much apart from a big plant and another building. Try and stay on a higher level as think the views to the sea would be lovely from there. In the main the staff were friendly apart from one female member of staff. We used the bar and sat at the terrace - a lot of flies about! The barman was super-friendly and couldn't do enough for us. The pool is large and good for swimming. From the secure back gate you come out onto the promenade and beach. Lots of restaurants along the promenade. The breakfast was great with a wide choice of food (and fizzy wine!) including made to order omelettes, fried eggs and pancakes.
- Official Description (provided by the hotel):
- A superior 4 star Hotel where the functional distribution of its spaces, with the sea as its protagonist, the decoration, with its strong personality, have all come together to give you an elegant, classical and functional Mediterranean Hotel. Located on the seafront, MS Amaragua looks out directly on to the Mediterranean and the pedestrian promenade of the traditional, infamous neighbourhood of the Carihuela, where the cultural cuisine of Pescaito frito (little fried fish) reaches its maximum expression. Only a couple of minutes walk away is the famous Benalmádena Marina, Puerto Marina. We have 275 rooms; of these 2 are luxury suites, 8 suites, 6 junior suites and 12 superior doubles. Restaurant, snack bar, swimming pool, lobby bar, conference and conventions halls, parking with 217 spaces, big outside swimming pool for adults and children, ample sun terrace, beach with direct access from the Hotel and the spa & fitness centre. 24 hour reception, press, left luggage, bellboy, room service, laundry service and rental service of bicycles, cars and motorbikes. CATERING To start the day, the Hotels restaurant, offers a complete and varied breakfast buffet with panoramic views of the Mediterranean . Enjoy the luminosity and relaxation at the swimming pool Bar La Ola, where Mediterranean cooking fuses with local cuisine and the after dinner drinks at sunset.Mens sana in corpore sano in the Health and Beauty Centre (SPA & Beauty Farm) the Hotel has at your service. The Hotel has on offer for its clients, an indoor climate controlled swimming pool, Jacuzzi, sauna, Turkish bath, gym, massages and an ample array of aesthetic treatments. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Expedia, Travelocity, getaroom.com, Despegar.com, Priceline, Hotels.com, Odigeo and Tingo so you can book your SENTIDO Amaragua 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:
- Amaragua Hotel Torremolinos
- SENTIDO Amaragua Torremolinos, Costa Del Sol, Spain


text:
Getting Started with Heartbeat
The third configuration file is the haresources file. Before configuring it, you need to do some housecleaning. Namely, all services that you want Heartbeat to manage must be removed from the system init for all init levels.
On Debian-style distributions, the command is:
/usr/sbin/update-rc.d -f <service_name> remove
Check your distribution's documentation for how to do the same on your nodes.
Now, you can put the services into the haresources file. As with the other two configuration files for Heartbeat, this one probably won't be very large. Similar to the authkeys file, the haresources file must be exactly the same on every node. And, like the ha.cf file, position is very important in this file. When control is transferred to a node, the resources listed in the haresources file are started left to right, and when control is transfered to a different node, the resources are stopped right to left. Here's the basic format:
<node_name> <resource_1> <resource_2> <resource_3> . . .
The node_name is the node you want to be the primary on initial startup of the cluster, and if you turned on auto_failback, this server always will become the primary node whenever it is up. The node name must match the name of one of the nodes listed in the ha.cf file.
Resources are scripts located either in /etc/ha.d/resource.d/ or /etc/init.d/, and if you want to create your own resource scripts, they should conform to LSB-style init scripts like those found in /etc/init.d/. Some of the scripts in the resource.d folder can take arguments, which you can pass using a :: on the resource line. For example, the IPAddr script sets the cluster IP address, which you specify like so:
In the above example, the IPAddr resource is told to set up a cluster IP address of 192.168.1.9 with a 24-bit subnet mask (255.255.255.0) and to bind it to eth0. You can pass other options as well; check the example haresources file that ships with Heartbeat for more information.
Another common resource is Filesystem. This resource is for mounting shared filesystems. Here is an example:
The arguments to the Filesystem resource in the example above are, left to right, the device node (an ATA-over-Ethernet drive in this case), a mountpoint (/opt/data) and the filesystem type (xfs).
For regular init scripts in /etc/init.d/, simply enter them by name. As long as they can be started with start and stopped with stop, there is a good chance that they will work.
Listings 4 and 5 are haresources files for two of the clusters I run. They are paired with the ha.cf files in Listings 2 and 3, respectively.
Listing 5. A More Substantial haresources File
deimos \ IPaddr::192.168.12.1 \ Filesystem::/dev/etherd/e1.0::/opt/storage::xfs \ killnfsd \ nfs-common \ nfs-kernel-server
The cluster defined in Listings 2 and 4 is very simple, and it has only two resources—a cluster IP address and the Apache 2 Web server. I use this for my personal home Web server cluster. The servers themselves are nothing special—an old PIII tower and a cast-off laptop. The content on the servers is static HTML, and the content is kept in sync with an hourly rsync cron job. I don't trust either "server" very much, but with Heartbeat, I have never had an outage longer than half a second—not bad for two old castaways.
The cluster defined in Listings 3 and 5 is a bit more complicated. This is the NFS cluster I administer at work. This cluster utilizes shared storage in the form of a pair of Coraid SR1521 ATA-over-Ethernet drive arrays, two NFS appliances (also from Coraid) and a STONITH device. STONITH is important for this cluster, because in the event of a failure, I need to be sure that the other device is really dead before mounting the shared storage on the other node. There are five resources managed in this cluster, and to keep the line in haresources from getting too long to be readable, I break it up with line-continuation slashes. If the primary cluster member is having trouble, the secondary cluster kills the primary, takes over the IP address, mounts the shared storage and then starts up NFS. With this cluster, instead of having maintenance issues or other outages lasting several minutes to an hour (or more), outages now don't last beyond a second or two. I can live with that.
2 Nodes configured, unknown expected votes
Hi, this article is easy to configure heartbeat.
my monitoring command showing
Last updated: Wed Mar 17 18:44:42 2010
Stack: Heartbeat
Current DC: node1 (e9de9b67-cebf-4dd6-aeab-0276b49320ed) - partition with quorum
Version: 1.0.5-3840e6b5a305ccb803d29b468556739e75532d56
2 Nodes configured, unknown expected votes
0 Resources configured.
Online: [ node2 node1 ]
Why am getting "unknown expected votes"
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Some of them talked for way too long before figuring it out.
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Episode 4.03 - Personal Effects
All hands are on deck to prevent a small town from being wiped off the map when a box of artifacts is accidently unleashed.
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Pete and Claudia reunite with Hugo to track down a deadly artifact at his nephew's prep school; Myka and Steve investigate an artifact that is killing a musician in New Orleans.
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Pete and Myka investigate a hockey player who is miraculously recovering from his injuries; Claudia is invited to a mysterious lunch with Mrs. Frederic; Artie and Steve has discovered that someone is stealing artifacts from the warehouse.
Episode 4.06 - Fractures
The team discovers someone has stolen Lewis Carroll's mirror from the warehouse.
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Gallery: Inflatable Research Lab Rolls Across the Arctic Tundra
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text:
Masters Degree in Film: Program Overviews
Get information on two options for master's degree studies in film. Read on to find out about admission requirements, coursework and career options in these fields of study.
Essential Information
Two program options are available to students interested in earning a master's degree in film. For each program, applicants must hold a bachelor's degree in a liberal arts or humanities field.
A Master of Arts in Film Studies program focuses on the theoretical and historical aspects of the field. Students learn to think and write critically about films, looking at areas such as film theory, comedy, documentary filmmaking and American cinema.
In a Master of Fine Arts in Film Production program, students gain practical skills in filmmaking and film design. Courses cover all aspects of filmmaking, including screenwriting, cinematography and editing. Applicants may have to submit a sample film for admission to the program, and a final thesis film is often required for graduation.
Master of Arts in Film Studies
A master's degree in film studies is synonymous to a master's degree in cinema studies. Students enrolled in a Master of Arts in Film Studies program learn about film theory and film history. Courses train students to think analytically and critically about films, as well as consider the philosophical and political implications of a film genre or a director's body of work.
Educational Prerequisites
Applicants to a master's degree program in film studies should hold an undergraduate degree in a liberal arts discipline; it's not necessary for applicants to have an undergraduate degree in film studies. Many universities also require applicants to submit scores for the Graduate Record Examination (GRE) and include letters of recommendation. A writing sample in some area of film or television criticism must also be submitted.
Program Coursework
Classes for the M.A. program introduce students to the academic study of film. Many of the courses require students to write papers that analyze film sequences or entire films. Specific course topics may include:
- Film theory
- Film history
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- American cinema
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- Documentary film
Popular Career Options
Film studies is an academic field, although graduates may also go on to work in various professional practice roles as filmmakers or screenwriters. Common career options for graduates of a Master of Arts in Film Studies program include:
- Film critic
- Film curator
- Film preservationist
Continuing Education Information
A Ph.D. program in film studies is an option for students who want to continue their graduate studies. The degree program prepares students for careers as film educators at major colleges and universities.
Master of Fine Arts in Film Production
The Master of Fine Arts in Film Production is the terminal arts degree for students interested in filmmaking. Part of the curriculum is devoted to film theory and history, but students mainly focus on developing skills in aspects of moviemaking. All aspects of production are covered, and students gain experience in screenwriting, editing, cinematography and directing before selecting a concentration. Students must generally complete a number of short films throughout the program, but a final thesis film is typically required to complete the requirements of the degree.
Educational Prerequisites
Applicants generally hold an undergraduate degree in some area of the humanities; a degree in film theory or film production is not required. The primary component of the application, however, is the portfolio. Portfolio requirements vary, but many film production departments request a short film that demonstrates an applicant's competence and familiarity with the medium. Letters of recommendation may also be requested.
Program Coursework
Hands-on experience is emphasized in the MFA curriculum. Students learn every aspect of film production by completing a number of short projects. Typical courses in this program often include:
- Screenwriting
- Cinematography
- Flatbed editing
- Producing
- Sound
- Film history
- Digital editing
- Film composition
Popular Career Options
Competition for film production jobs is fierce. Graduates typically gain experience in lower-level jobs and work their way up. Alternatively, some grads choose to teach at the community college level. Common job titles for a graduate of this program include:
- Grip
- Camera operator
- Assistant director
- Cinematographer
- Community college instructor
- Film producer
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text:
Hey guys,
I'm wondering if is there a way to know when the user clicked the first and second time on link?
Link
And When the user clicks the first time do a function and when the user clicks the second time open the link url.
I'm trying to make a dropdown menu work on touch devices.
If you know another way to make it work on touch devices let me know please
Just set a class on the first click:
$(document).ready(function() { $("a").click(function() { if(!$(this).hasClass('clicked')) { //place code for first click $(this).addClass('clicked') //open link next time return false; //prevent from opening link this time } }); });
That's a smart idea, Thanks RikdeVos
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text:
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This article appeared in a journal published by Elsevier. The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
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Computational Biology and Chemistry 33 (2009) 196–204
Contents lists available at ScienceDirect
Computational Biology and Chemistry
journal homepage: www.elsevier.com/locate/compbiolchem
Research Article
Search of regular sequences in promoters from eukaryotic genomes
Andrew Shelenkov∗, Eugene Korotkov
Bioengineering Centre of Russian Academy of Sciences, 117312 Moscow, Pr-t 60-tya Oktyabrya, 7/1, Russian Federation
a r t i c l e i n f o
Article history:
Received 26 June 2008
Received in revised form 8 February 2009
Accepted 18 March 2009
DNA sequence analysis
Protein binding sites
a b s t r a c t
This notion expands the "latent periodicity" term. The novel method for revealing regularity based on the
runs test is described. The search of regular sequences in eukaryotic promoters has shown that more than
60% of them possess a regularity property on statistically significant level. Possible biological functions of
regularity are discussed together with the possibility of using this characteristic for performing promoter
© 2009 Elsevier Ltd. All rights reserved.
1. Introduction
At the present time, a wide-scale analysis of DNA sequences
from various genomes, including human ones, takes place. One of
the most important goals of this analysis is a characterization and
determination of the functions of various genes. During the last
decade, several reliable methods of predicting protein-coding DNA
regions have been proposed (Claverie, 1997). However, the predic-
tion of regulator regions, in particular, promoters, still remains a
challenging task although different methods have also been pro-
posed for their prediction (Bajic et al., 2002; Davuluri et al., 2001;
Ohler et al., 2002). Promoter is a genome region located near the
site of transcription initiation and playing the key role in genetic
regulation (Pedersen et al., 1999). Promoters receive signals from
various sources (e.g., from cell receptors) and control the level of
transcription initiation that to a great extent determines a gene
expression (Dieterich et al., 2005). Thus the promoter revealing is
an important step in performing gene annotation.
To distinguish genome regions containing and not containing
promoters (the latter, obviously, being the most part), a large set
of characteristics was used, for example, CpG islands (Davuluri et
al., 2001; Bajic and Seah, 2003), TATA-boxes (Ohler et al., 2002;
Knudsen, 1999), CAAT-boxes (Ohler et al., 2002; Knudsen, 1999),
some typical sites of transcription factors' binding sites (Ohler
et al., 2002; Knudsen, 1999; Solovyev and Shahmuradov, 2003),
pentamer matrices (Bajic et al., 2002), oligonucleotides (Scherf et
al., 2000), and also combined approaches were used (Xie et al.,
∗Corresponding author. Tel.: +7 499 135 2161; fax: +7 499 135 0571.
E-mail address: firstname.lastname@example.com (A. Shelenkov).
2006). Besides, some pattern recognition techniques were used
such as neural networks (Ohler et al., 2002; Bajic and Seah, 2003;
Knudsen, 1999), linear and discriminant analysis (Davuluri et al.,
2001; Solovyev and Shahmuradov, 2003), interpolation Markov
model (Ohler et al., 2002), analysis of independent constituents
(Matsuyama and Kawamura, 2004) and some other methods
(Gershenzon et al., 2006). However, the analysis of experimental
data has shown (Bajic et al., 2004) that the issue of choosing sta-
tistically significant biological signals to be used in the promoter
prediction programs still remains unsolved. There is no character-
istic which describes the whole variety of promoters, and each of
the features revealed during examination of promoter sequences
has its own usage limitations (Xie et al., 2006). Therefore, there is a
need for finding some new characteristics which would be specific
for the promoters, but at the same time would be quite flexible to
meet the variety of their types.
The presence of periodicity and other structure elements in
genetic texts was previously revealed by various mathematical
methods (Benson, 1999; Konopka, 1994; Konopka and Martindale,
1995). In this paper we introduce a new term – regularity – to char-
acterize a sequence and propose the method of identification of
regular sequences. We apply the method developed to search the
regular sequences in promoters from various eukaryotic genomes.
The notion of regularity is an extension of the "latent periodic-
ity" term (Korotkov et al., 2003; Laskin et al., 2005; Shelenkov
et al., 2006, 2008) for the case when the positions of individual
nucleotides in a period are not fixed rigidly. Regularity is a non-
sequence being examined. To reveal the regularity we divide the
sequence into equal-sized intervals (or "periods") and calculate
the degree of "non-randomness" of each nucleotide distribution in
1476-9271/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
Page 3
Author's personal copy
A. Shelenkov, E. Korotkov / Computational Biology and Chemistry 33 (2009) 196–204
these periods. We used the runs test (Hoel, 1966; Brownlee, 1965)
as a criterion for estimating the non-randomness of these distribu-
tions. The method of calculating the number of runs is illustrated
below. The definition of a regular sequence and calculation of a
quantitative measure of the level of sequence regularity are given
in Section 2.1.
The method described in the current paper can be used for
their structural composition is similar to the one of periodical
sequences. The main advantages of the method are its comparative
ment of the period type (e.g., in a form of a frequency matrix), and,
being the most important, the possibility of revealing the sequence
regularity at simultaneous presence of nucleotide indels and sub-
stitutions in the analyzed sequence.
In Section 3 it is shown that the majority of known promoters
possess the regular structure.
2. Methods and Algorithms
2.1. Regularity Definition and Its Quantitative Measure defined
Using Runs Test
Let us define the regular sequences as DNA sequences which
have the same (or close) number of certain type (or all) nucleotides
in each period, while nucleotides are distributed in a same way
along the periods. Here the periods of length n are the DNA frag-
ments of the same length located one after another in a sequence.
Let us consider the sequence S=s1, s2, ..., sL, containing the char-
acters from the alphabet Q={a, t, c, g}. In Fig. 1, DNA sequence is
we place F at 0 and L+1 positions of S. Then we count n nucleotides
Fig. 1. Application of the runs test to determination of adenine distribution "non-
randomness" along the periods of the length 3 nucleotides. The sequence under
study is divided into separate periods by insertion of the symbol F after each three
nucleotides starting from the zero position of the sequence. Then the sequence of
categories (or codes) is formed by changing the symbol F to 0 and the symbol 'a' to
1, and the number of runs is calculated. Run is a sequence of identical elements that
is preceded and followed by different elements or no element (the beginning or the
end of the sequence). For example, the sequences of codes 000111 and 01 contain
to the one in non-periodic sequence (A). This allows using the number of runs as
periodicity measure which is almost insensitive to nucleotide indels. This is shown
in (C), where the deletion of 'c' from 9th position of the periodic sequence is made.
It is clear that the number of runs does not change and remains equal to17.
starting from the first position of S and place another symbol F.
We repeat this process till the end of the sequence S. As a result,
we obtain the sequence S?=Fs1, s2, ..., sn, Fsn+1, sn+2, ..., s2n, F,
..., sLF (the length of the last period can be smaller than the gen-
eral length). Practically, the presence of regularity means that the
distribution of positions of some nucleotide along the sequence is
the same as the distribution of the positions of symbol F along this
sequence. We use the runs test to define the quantitative measure
of the regularity.
Runs test (Wald–Wolfowitz test) (Hoel, 1966; Brownlee, 1965)
is a non-parametric test that is used to test the hypothesis H0stat-
ing that two (or more, generally) datasets represent the random
independent samples with amounts n1and n2(n2+n2=N) from
the same universal set, i.e., the distribution functions of these sam-
ples are the same. In use of the test, it is estimated the number of
runs in a series which elements may generally possess k different
values. Let us consider the case k=2. Observation results are writ-
ten as the static series of joint sample T, while the data belonging
to a certain group are given by the coding variable that may pos-
sess two values (0 and 1, where 0 shows the value belonging to the
first sample, while 1 to the second one). The values of coding vari-
able form the series R that is called "the sequence of categories" or
"the sequence of codes". To apply the runs test, the elements of the
series T are sorted in ascending order with making simultaneous
rearrangements in the series R.
Run is a sequence of identical elements that is preceded and
followed by different elements or no element (the beginning or
the end of the sequence). The test statistic is a number of runs
in the sequence of codes. If the hypothesis H0is true, then both
samples should be mixed well in the static series and thus the
number of runs should be large. If the samples have been derived
from the universal sets having different distributions (which vary
in mean value or dispersion), then the number of runs will be
For example, if the sequence S looks like tcgcgcattattcaagtacc,
'a' is {0, 0, 1, 1, 0, 1, 1, 0, 1, 0} (runs are underlined). Here the value
in the sequence of codes corresponding to the symbol F is 0, and
the one corresponding to the nucleotide 'a' is 1. In this example the
number of runs is 7.
To introduce the quantitative measure for a case of nucleotide
sequences, we derive four sequences of codes K(i) from the
sequence S?. The sequence of codes K(i), i=1, 2, 3, 4 is derived from
1, while all other symbols are ignored. At that it is considered that
the properties of a source sequence are represented by the proper-
ties of binary sequences (Lobzin and Chechetkin, 2000). Here q(i) is
a symbol from the alphabet Q defined above. Actually, this means
that we compare the samples containing the position numbers of
F and q(i) in the sequence S?. We calculated the number of runs
r(i) for each introduced sequence of codes. Statistical significance
modeling (Monte Carlo method). It included performing a random
shuffling of the symbols of initial sequence S, then the sequence
S?was built for the newly obtained sequence together with the
sequences K(i), and the numbers of runs r(i), i=1, 2, 3, 4 were deter-
mined. As a result, P values for each of the number of runs r(i) were
obtained for random sequences (we used P=100). After this, the
mean value ?r(i) and the standard deviation ?r(i) were calculated
for four sets of the obtained values. The formula for calculating the
statistical significance Z(i) of the number of runs r(i) was:
Z(i) =r(i) − ?r(i)
Page 4
Author's personal copy
A. Shelenkov, E. Korotkov / Computational Biology and Chemistry 33 (2009) 196–204
It should be noted that the runs test can perform the reli-
able comparison of the samples comparable by their amounts
imately equal to the number of the nucleotides of each type. If
the number of the nucleotides of some type is much larger or
smaller (more than two times) than the number of the sym-
bols F, then the application of the runs test may not reveal the
sequence regularity. Let us modify the example given above. Let the
sequence S=tcgcgaaaattaaacaaaag, S?=FtcgcgFaaaatFtaaacFaaaagF.
In this case the static series for the nucleotide 'a' looks like {0, 0,
1, 1, 1, 1, 0, 1, 1, 1, 0, 1, 1, 1, 1, 0}. This example shows that the num-
ber of runs does not increase when the number of symbols in each
period increases. For long periods (>4) this fact can be the cause of
not revealing some regular sequences.
To solve this problem, we inserted additional symbols F into the
sequence S?. In this case the periods of a sequence S are divided into
parts (or ?boxes?, analogously to the models used in combinato-
rial calculus) each in the same way. For the example given above
the sequence S?after inserting an additional symbol F into each
period may look like FtcgFcgFaaaFatFtaaFacFaaaFagF (new sym-
bols are shown bold-faced). We considered all possible placements
of new symbol and chose the position (identical in each period)
for which the number of runs was the greatest. Then a statistical
significance was calculated again using Monte Carlo method as it
period giving the greatest number of runs was determined for each
sequence obtained by random shuffling of the initial sequence S.
Thus we determined the maximal number of runs rp(i) where p
shows the order number of a random sequence. We shuffle the ini-
tial sequence 100 times, so p=1, 2, ..., 100. Then the mean value
?rand the standard deviation ?rwere calculated for the set of 100
values and the corresponding value Z1was calculated by formula
(1) (here the index shows the number of additional symbols F in a
sequence S?).
To simplify the method description, we discuss the calculation
only for one nucleotide type. Evidently, the calculations were per-
formed in the same way for each type.
If m(q(i))−m(F(i))>L/n (i.e., numbers of additional and original
symbols are comparable), then one more symbol F was added to
each period in the same way as it was described above. Thus the
value Z2was calculated. Here m(q(i)) shows the number of type i
nucleotides, m(F(i)) shows the number of symbols F in a sequence
S?for this nucleotide. Upon completion of symbol addition process,
we have the set of values Z0, Z1, Z2, ..., Zk. We choose the maximal
one from these values –let us denote it Z.
The large positive value of Z means that the observed number
of runs greatly exceeds the expected one, i.e., the average length
of the run in static series is close to 1. This fact witnesses that the
number of times the symbol under study occurs in "boxes" (or in
plain periods) are similar on statistically significant level. In a case
of large negative value, the presence of a symbol in "boxes" differs
from period to period, so artificial symbols and nucleotides are not
"mixed" well. In this work, we focus on the large positive values
since they suppose that the source sequence possesses the peri-
odicity (possibly, the fuzzy one) or, generally, that the sequence is
All sequences described in Section 3 are regular. We chose this
term because these sequences possess some regular structures
which are represented by the regular occurrence of nucleotides
inside the "boxes" limited by the symbols F. Such regularity of
nucleotide distribution in "boxes" has a statistically significant dif-
ference from a distribution of symbols in random sequences. This
fact is reflected by the large values of corresponding Z variables.
However, not all of these sequences are periodic in the traditional
way. In fact, most of them are not, this is why the present methods
of revealing periodicity are not able to find them.
2.2. Calculation of Joint Statistic and Regularity Search
We have considered above the application of the runs test for
searching the regularity of one nucleotide in the sequences under
study. However, to perform the deeper comparative analysis of
the sequences it is necessary to estimate regularity for all four
nucleotides simultaneously. To do this we first calculate the statis-
tic Z (formula (1)) for each nucleotide separately. Let us designate
the values obtained for a, t, c and g as Za, Zt, Zc and Zg, respec-
tively. All these variables have a distribution close to normal one
as it will be shown below. Let us use the property of normal distri-
bution to get the new variable whose distribution is also normal.
We obtain:
Zsum=Za+ Zt+ Zc+ Zg
=Za+ Zt+ Zc+ Zg
n was chosen and four sequences S?(one for each nucleotide) were
built for a sequence S in a window by introducing the symbols F. n
belonged to the range 2–16. For each of these four sequences the
the value Zsumwas calculated for the obtained values of Za, Zt, Zcи
Zgby using formula (2).
We tried to obtain the most exact borders of the subsequence to
make easier the future investigation of its function. So the search of
the regular subsequence with the maximal value of Zsum within
a window was performed by changing the border of the ana-
lyzed sequence, namely, its left and right borders were changed
independently with a step=2 and a statistic was calculated for
each such subsequence. Thus, the subsequences of the given win-
dow having length from 50 to 500 nucleotides were considered.
Results included all non-overlapping subsequences of the window
for which the value of Zsum exceeded the threshold. The subse-
quences having length less than 50 were not considered because
the sample size in this case was not large enough to get the reliable
results at the chosen level of statistical significance.
Parameters of sequence scanning (window=500, step inside a
window=2) have been chosen, on the one hand, to provide the
reasonable rate of calculations and, on the other hand, to reveal all
which possess regularity not only for all four nucleotides, but
also for all their subsets (including regularity for the only one
nucleotide), we calculated the values of the statistic for all pos-
sible combinations of nucleotides for the considered subsequence
among them.
If we consider only the statistic for all four nucleotides, then
the negative value for one nucleotide can decrease the value of
Zsumbelow the threshold. For example, for the case Za=−4, Zt=3,
Zc=3 and Zg=3 we obtain Zsum=2.5, whereas Ztcg=5.2, i.e., there is
a regularity for three nucleotides. Thus performing the calculations
for all nucleotide combinations allowed lowering the influence of
negative statistic values for some nucleotides on the possibility of
regularity revealing.
2.3. Building of the Regularity Scheme
It is evident that the use of statistical significance Z as the
only characteristic of regular sequences does not allow revealing
regularity types and comparing them. Yet such a characteris-
tic is necessary, especially for investigating the biological role of
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regularity. We propose to use the regularity scheme as such a char-
arrangement in the sequence for which the nucleotide distribu-
tion occurred to be the "mostly non-random", that is, for which
the value of Zsumwas maximal, e.g., the scheme given below shows
sequence S?having nucleotide "a" in any period position from 1st to
"c" may occur in 1st–3rd and 4th–6th positions of the period, etc.
In this case there exists regularity for three nucleotides—"a, c, g",
while the period length equals 6 nucleotides.
2.4. Distribution Density of Z for Random Sequences. Choosing
the Threshold Value of Zsumfor Searching Regular Sequences in
The conformity of results obtained by using a simulation mod-
eling is necessary to be checked by applying the method used to
ing regularity in a random sequence on significance level higher
than a threshold will tend to zero. Besides, building the specter of
of Zsum.
The specter of statistic Z for one nucleotide was built for the
sequence generated by the random number generator (period of
the generator was ∼2×1018) for the period lengths from 2 to 16
nucleotides. The length of the sequence was 10 million nucleotides
(i.e., approximately 10 times larger than the total length of the
analyzed promoter sequences). The initial frequencies of each
nucleotide's occurrences were equal to the ones in the analyzed
set of promoters. The generated sequence was analyzed by using
a scanning window as it was described above. For example, the
specter has been obtained for the period lengthn=4. Mean value of
were not found. The number of Z values greater than 4.0 was 3, the
maximal value was 4.1982. The results obtained for other period
lengths were analogous.
In addition, the specter of a joint statistic Zsumwas built for the
using the normal approximation for its calculation. A joint statistic
was calculated for all four nucleotides and for all possible combi-
nations of two and three nucleotides (i.e., in the same way as it had
been done for searching the regularity in promoters). Distribution
of the statistic values in all cases is analogous to the one obtained
for single nucleotides.
one random sequence will be found to be regular for the length of
1 million nucleotides (the total length of the promoters analyzed).
To get more accurate estimation, we performed the same cal-
culations for the random sequence having the length equal to 100
million nucleotides. In this case 39 regular regions with Zsum≥4.0
results we can also conclude that not more than one DNA sequence
ers while using our algorithm with a threshold value Zsum=4.0. Let
us also note that there were no sequences revealed with Zsum≥5.0.
So, we consider a DNA sequence from the analyzed set of pro-
moters to be regular if statistic value for it is Zsum≥4.0. Because
of the reasons stated in this chapter, we believe this value to be
relevant for the real data.
2.5. An Algorithm of Filtering the Results
When changing the window borders in the range 50–500 with
the step=2, it is possible to reveal the sequences possessing a regu-
Such an overlap (either full or partial) may occur when the same
sequence was revealed at different scanning window positions. To
prevent this, we leave only the sequence having the greater value
of Zsum if some sequences overlapped each other by more than
n in DNA sequences. However, the fact that Zsumfor some sequence
is greater than the threshold for some period length n does not
allow making an unambiguous conclusion regarding the presence
of regularity exactly for this length. The case is that the regularity
for the length n may be caused, for example, by the presence of
regularity for multiple periods, i.e., for the period lengths nk, where
k=2,3,.... In this case the period length n may give the statistical
significance greater than threshold, while the real regularity occurs
for the lengths kn. We will consider that there is a regularity with a
period length n in DNA sequence if the value of Zsumfor this length
is greater than the threshold and the values of test statistic for all
other analyzed period lengths are smaller (they may either exceed
the threshold or not). Thus we calculated the value of Zsumfor all
period lengths in a range from 2 to 16 nucleotides for the sequence
length n, then the sequence was excluded from the consideration.
In addition, we performed one more filtration step to provide
the maximal reliability of the results. For each of the sequences
revealed to be regular we built the information decomposition
specter (Korotkov et al., 2003) which shows the statistical signif-
icance of the found period expressed in the terms of Z (formula
(1)) against the period length. Information decomposition reveals
the periodicity without indels in nucleotide sequences, but it can
also reveal the latent periodicity (Korotkov et al., 2003). Let the
period having the maximal value of Z be referred as the "maximal
period". We will consider that the maximal period really exists in
a sequence if Z≥4.0 for this period in information decomposition
specter. In case when such maximal period existed in a sequence
and its length did not correspond to the length of regularity n, we
excluded the analyzed DNA sequence from consideration because
it was most likely that this length of regularity did not correspond
to the maximal value of Zsum.
The set of sequences passed through all steps of filtration was
considered as the set of final results.
2.6. Choosing of Promoter Sequences to be Analyzed for Regularity
The source promoter sequences were obtained from EPD
(Eukaryotic Promoter Database) (Schmid et al., 2006), version 93
preliminary data, was 4809). We have chosen 2236 sequences rep-
resenting all groups of organisms. To do this, we used the database
option "representative set of not closely related sequences". The
use of this option ensures that the set of selected sequences will
not contain any 2 sequences having the pairwise similarity of
more than 50%. The range of promoter regions was (−499, +1)
where +1 corresponded to the transcription initiation site. Since
we were searching the regularity only in promoter sequences,
that is, not including the genes, the analyzed set of promoters
included 2236 sequences, each of the length equals to 500. For
each of these sequences the calculations described above were per-
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Fig. 2. An example of the periodic sequence revealed by the method of information decomposition which was also revealed as regular. Here the periodic sequence (shown
in bold) is a part of regular sequence (A). An alignment of the modified and initial sequences. Similarity level is 61.4% (B).
3. Results
3.1. Search of a Regularity in Artificial Periodic Sequence with
Let us consider an example of regular nucleotide sequence. The
method we developed reveals regularity on statistically significant
level in it, while other methods (e.g., Korotkov et al., 2003; Benson,
form (Sharma et al., 2004), are not able to reveal the periodicity.
the period length=5 was chosen as an initial one. It contains 80
copies of the {attcg} subsequence. Then indels and substitutions
simulating mutations were made in it by using a random number
lated for n=5. Upon making 80 indels, the following sequence has
been obtained (length=399):
An alignment of the initial and the newly obtained sequences
is shown in Fig. 2B. The value of the test statistic for this new
sequence was Zsum=5.02 (Za=3.41, Zt=0.92, Zc=2.49, Zg=3.21). At
the same time, the methods based on Fourier transform (Sharma
et al., 2004), information decomposition (Korotkov et al., 2003)
and dynamic programming (Benson, 1999) have not revealed the
periodicity with the period length n=5 in this sequence on sta-
tistically significant level. Thus the method we developed is less
3.2. Regularity Search in Sequences Possessing the Latent
Periodicity Revealed by Information Decomposition Method
We have also studied the possibility of revealing regular sub-
sequences by using our method in the DNA sequences with latent
periodicity revealed by the method of information decomposition
(Korotkov et al., 2003). To do this, we performed the search of the
latent periodicity on the same set of promoters by using the infor-
mation decomposition. The total number of periodic sequences
with a period length in a range from 2 to 16 was 109, while 62
of them had their length greater than 50 nucleotides (the mini-
mal length for revealing regularity). All these sequences have been
revealed as regular with a length of regularity corresponding to the
length of the latent period. Therefore, in this case the regularity
search reveals the sequences possessing the latent periodicity.
An example of the sequence revealed by both methods is shown
in Fig. 2A.
3.3. An Example of a Regular Sequence Found in a Promoter
Let us give an example of a regular sequence revealed in a
promoter. The regularity with length of 10 nucleotides has been
revealed in locus EP77531 in the region (−113, −44). The test statis-
tic for it was Zsum=5.1 (Za=1.9, Zt=3.1, Zc=2.0, Zg=3.1). The DNA
sequence looked like:
sequences S?for the nucleotides a, t, c and g were:
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Table 1
The number of regular sequences found in promoters for the period
lengths 2–16.
Period length The number of regular sequences
It should be noted that no value of statistical significance Z for
single nucleotides exceeds the threshold value, that is, regularity is
a joint property of all four nucleotides of a promoter sequence.
3.4. Results of Searching Regular Sequences for a Period Length
2–16 in Promoters
We searched for regular subsequences in promoter sequences
from the EPD (Schmid et al., 2006) using the following scanning
parameters: window size=500, step for window border vari-
ation=2. The length of the regular subsequences revealed lay
in a range 50–500. The data regarding the number of revealed
sequences are given in Table 1.
We have analyzed 2236 promoters totally, while regularity has
been revealed at statistically significant level in 1342 of them. Thus
more than 60% contain the regular subsequences with a period
length range from 2 to 16 nucleotides. The regularity has been also
revealed in other promoter sequences, but the level of statistical
tribution of the length of regular subsequences (Table 2) and their
arrangement in promoters.
The longest regular sequence had a length 484bp. However, as
it can be seen from the Table 2, the majority of the sequences had
sequences we selected the ones having the greater statistical sig-
nificance, not length (i.e., we did not try to maximize the length of
the sequence revealed), such length distribution is quite expected.
Such a selection process allows setting of the more exact regularity
Table 2
Length distribution of the regular sequences revealed.
Length range, base pairs. The number of the found sequences
Investigation of regular sequence arrangement in promoters is
more important for understanding the biological role of the reg-
ularity than sequence length distribution. We divided a promoter
sequence into the intervals of length equal to 10bp and counted
the number of sequences that fall into each of these intervals. Since
the minimal length of regular sequence was 50bp, it is evident that
each sequence fell into several intervals. First of all, we wanted to
know if the distribution of regular sequences along the promot-
ers was random. If such a distribution has maxima or minima on
the certain regions of promoter, there is a possibility of connect-
ing the regularity to some biological function which these regions
We used the simulation modeling (Monte Carlo method) to
determine if the regular sequence distribution was random. To
do this, we randomly placed all the regular sequences that were
found along the promoter sequence. At this, the lengths of reg-
ular sequences corresponded to the ones in the set of revealed
sequences, e.g., if there had been 22 revealed sequences with the
length 96, then the same number of sequences having this length
were randomly placed. Practically, we used a random number gen-
erator to determine only the starting point of the sequence. Its
coordinate might vary in a range (1, 500-k), where k is the length of
promoter borders. Such placement of sequences was repeated 200
times, and after each iteration the number of sequences fallen into
the intervals with length 10 was determined. Based on these data,
the mean value and the dispersion were calculated, and then the
statistical significance Z was calculated by using the formula anal-
ogous to (1). The values of Z obtained for each interval are shown in
Fig. 3. As shown in the figure, the number of the regular sequences
revealed in the interval (−99, +1) relative to the beginning of a gene
considerably increases the number expected for the random place-
Z exceeds 5.0. Besides, the declination of Z in the negative direction
was observed in the intervals (−389, −169). We think that this fact
Fig. 3. Decline of the number of regular sequences from expected one for the random distribution, in magnitude of standard deviation. The right borders of the intervals are
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A. Shelenkov, E. Korotkov / Computational Biology and Chemistry 33 (2009) 196–204
is caused by the prevalent localization of the regular regions in the
interval (−99, +1).
4. Discussion
Currently high emphasis is placed on the investigation of pro-
moter sequences since they determine gene activity in eukaryotic
and prokaryotic cells. If it is possible to classify promoters by some
quantitative sequence properties and, after that, to find a connec-
tion between these properties and the gene expression in certain
cells or at the certain moment of organism development, then this
1997; Werner, 2003). On the other hand, the improvement of algo-
in eukaryotic genomes more accurate (Fickett and Hatzigeorgiou,
revealing of promoter region points to the beginning of a gene. It is
important to develop new mathematical approaches to solve these
problems, which may reveal the new rules of promoter sequence
organization, and which will be used both for identification of pro-
moter sequences and for their classification. In the present work
we have introduced the "regularity" term and have found that reg-
ular sequences are mostly present in the region from −99 to +1
nucleotide relative to the site of transcription initiation. RNA poly-
merase usually binds with promoter at the region (−45, +5). Totally
some tens of proteins are involved in transcription complex forma-
tion. By the previously obtained data (Zhang, 2007), the promoter
region (−38, +5) is a binding site of TFIIB (−38, −32), TBP (−31,
−24), TFIIB (−23, −17) and TAF1 (−2, +5). The distribution of regu-
the region (−99, +1) is shown in Table 3. A regular sequence spans
several intervals. Each value of the Table 3 represents the number
of regular sequences of some period found in the given interval. For
example, the number of sequences with the period length equal
to 10 in the interval (−59, −50) was 60 (of course, each of these
sequences also appears in some other intervals since its minimal
length is 50). Comparing the previously obtained data with Table 3
and Fig. 3, we see that the binding regions of transcription factors
and RNA polymerase are entirely overlapped with the regions in
which the number of the regular sequences has exceeded expected
values. Based on this, we can suppose that the sequence regularity
may be connected with evolutional relationship of various tran-
scription factors that lead to the similarity of DNA sequences to
which these factors are able to bind. Such similarity may produce
regularity. The regularity in a region (−99, +1) has been revealed
on statistically significant level for approximately 60% of the pro-
moter sequences under study, while for other 40% it has also been
revealed, but the significance level was lower than the threshold
factors for different promoters. Different sets, in turn, may lead to
different statistical significances and regularity length in the region
(−99, +1).
Besides, the revealed regularity may be connected with a DNA
molecule bend in the region of RNA polymerase binding (Mizuno,
1987; Tchernaenko et al., 2008). The regularity of nucleotide inter-
change may be the cause of DNA bending formation with a possible
purpose of transcription complex formation facilitation (Ozoline et
al., 1999; Bolshoy and Nevo, 2000).
Periodicity of promoter sequences was previously investigated
by the methods based on Fourier transform (Ioshikhes et al., 1999;
Kutuzova et al., 1997, 1999; Bolshoy and Nevo, 2000). Results
obtained in the papers (Kutuzova et al., 1997, 1999) show that there
is a periodicity with a period length from 6 to 8 nucleotides in the
region (−99, +1). The authors suppose that the periodicity revealed
is mainly concerned with interaction of a DNA polymerase with the
region from −99 to +1 (core promoter). These conclusions are in
complete accordance with the data obtained in the present work.
In (Ioshikhes et al., 1999; Bolshoy and Nevo, 2000) periodicity of
promoter regions was also investigated by Fourier transform, as a
result of which the periodicity with a period length between 10
and 11 nucleotides has been revealed. The authors concluded that
such type of periodicity is specific for nucleosome binding near +1
position. It was also noted that this periodicity occurred because of
regular interchange of AA and TT dinucleotides (Herzel et al., 1999;
Cohanim et al., 2006; Salih et al., 2007). These data are also in good
accordance with the results obtained by us, since it is clear from
Table 3 that the main contribution to the observed regularity in the
interval (−99, +1) is made by exactly the period length from 10 to
oped reveals the regularity individually for each promoter, while
the use of Fourier transform can reveal periodicity with a period
length from 10 to 11 only for the certain set of promoter sequences
(Ioshikhes et al., 1999). This fact shows that our approach is more
sensitive and thus can be used for developing the recognizing algo-
rithms for individual promoter sequences.
Integrally, the fact that the largest part of regular sequences
in the region (−99, +1) possesses a regularity with a length
10–12 nucleotides suggests that the nature of regularity differs for
different regularity lengths. Comparatively short regularity (2–6
nucleotides), as it was noted above, may be important for bind-
Table 3
Distribution of regular sequences by the period length in the region (−99, +1).
Period length/interval
−99; −90
3 43
7 15
9 15
13 50
14 29
15 32
−89; −80
−79; −70
−69; −60
−59; −50
−49; −40
−39; −30
−29; −20
−19; −10
−9; +1
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in promoter region. Such a bend may provide the nucleosome posi-
tioning and/or facilitate the binding of RNA polymerase near +1
The testing of the developed mathematical approach has shown
that it was able to reveal the perfect and the latent periodicity
with comparatively small number of indels. However, the method
used could not find the regions where long nucleotide insertions
have occurred. In this case the sequences of codes K(i) may contain
rather larger number of empty "boxes" that will lead to substantial
decrease in statistical significance for such a region. This may be
the reason why the regularity in some promoter sequences in the
region (−99, +1) was revealed with Z<4.0.
Our method is not seriously affected by transpositions of neigh-
boring nucleotides. Within the limits of one "box" in a sequence of
codes K(i) the nucleotide position is not important for the method,
thus the transpositions of the type AT→TA and similar ones do not
influence the possibility of revealing the regularity in nucleotide
revealing the regions of protein binding to DNA since due to certain
conformational mobility of the proteins this process may require
the presence of certain nucleotides not in the specific promoter
1997; Werner, 2003). Such transpositions cannot be taken into
account by the methods based on dynamic programming during
making comparison of nucleotide sequences. The presence of the
pair AT against the pair TA in alignment will be considered as a lack
of similarity which will decrease the weight of final alignment.
The results obtained by us may be useful for identification
of potential promoter sequences in eukaryotic genomes. Ear-
lier multiple algorithms of searching and identifying promoter
sequences have been proposed (Pedersen et al., 1999; Fickett and
Hatzigeorgiou, 1997; Werner, 2003; Ohler and Niemann, 2001;
Anwar et al., 2008; Hutchinson, 1996; Prestridge, 1995; Reese,
2001). Most of these approaches are based on searching certain
motifs in DNA sequences, wherein the mathematical methods of
motif identification can be different. The common result of these
works is that it is possible to reveal the significant part of known
promoters (true positives), while the number of incorrect predic-
tions (false positives) remains rather high. Usually it is possible to
reveal more than 50% of existing promoters with a background
of one false prediction per about 1000 DNA bases (Fickett and
Hatzigeorgiou, 1997). We think that a regularity revealed by us can
lower the number of false positive predictions. To achieve this, we
should reveal regularity in all known promoter sequences and try
to classify the regularity revealed for each DNA base of each known
searching in genome sequences based on the classes obtained ear-
lier. The algorithms of promoter identification developed earlier
may be applied only to the DNA sequences in which the regularity
of certain class has been found. We suppose that such a combined
approach can significantly (by several times) reduce the number of
false positives which are revealed currently.
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