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IT TOOK 50 million years of continual shrinking to turn massive dinosaurs into the first flying birds.
"No other dinosaur group has undergone such a long and extended period of miniaturisation," says Mike Lee of the South Australian Museum in Adelaide. His team studied theropods, the two-footed dinosaurs like Velociraptor from which birds evolved. They tracked how 1549 skeletal features changed in 120 theropods over 50 million years.
They found 12 major evolutionary steps where theropod groups split to form new kinds of dinosaur. At each break, the ones that ended up evolving into birds shrank. They went from 238 to 0.5 kilograms, and changed four times as fast as other theropods (Science, doi.org/tx7).
Shrinking must have been beneficial, says Lee. "It would have permitted them to chase insects, climb trees, leap and glide, and eventually develop powered flight."
Being small may also have helped birds survive the dinosaur extinction, says Bhart-Anjan Bhullar of Yale University. It looks like the extinction "took out all landlocked animals above a certain size".
This article appeared in print under the headline "Shrink a bit more and add feathers"
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Red Carpet™ Barbie® - Blue Gown
Designed by: Bill Greening
Release Date: 6/2/2014
Product Code: BJV54
Barbie® doll's closet contains glam fashions for every event imaginable—from red carpet premieres to galas to the most amazing parties on the planet! Each doll in the collection features a beautiful gown, rooted eyelashes, dramatic earrings, a bracelet and a necklace or purse and shoes.
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For the adult collector.
Production doll may vary from the photo shown above. Mattel reserves the right to modify the fashion/fabrics, sculpt, hair color/style, and accessories. Doll cannot stand alone as shown.


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Doc Green and Steve Rogers go on an adventure!
As the world comes to an end, Widow flashing back to one of her first jobs for the Red Room.
How did the Ultrons rise to power?
Volume »
Banshee is a mutant with a sonic scream. Born in Ireland, he pursued a career in Interpol, the X-Men, Generation X as Co-Headmaster, and founded X-Corp. He is the father of Theresa Cassidy as well as Black Tom Cassidy's cousin.
Warlock is a member of the Technarchy race, and a New Mutant. He is considered a mutant because he can experience emotions.
Jean Grey was one of the five original X-Men. An omega-level mutant telekinetic and telepath, Jean has gained near limitless powers as a recurrent host of the Phoenix Force. She is known for her many returns from death and as the deceased wife of Cyclops.
Franklin is the son of Mr. Fantastic and Invisible Woman of the Fantastic Four. Franklin is an Omega-level mutant possessing nigh-omnipotent psionic powers and vast reality manipulating powers. Even at a young age he is one of the most powerful beings on Earth.
Susan Storm is a founding member of the Fantastic Four and later the Future Foundation. She is able to create invisible force fields of any shape she conceives and able to turn herself and anything she's in contact with invisible.
Cable is the time-traveling son of Cyclops. He was infected with the deadly techno-organic virus at a young age, which he kept at bay using his advanced psionic powers. He also raised the mutant messiah, Hope Summers.
Reed Richards, also known as Mr. Fantastic, is the leader of the Fantastic Four. He can stretch his body to great distances due to his exposure to cosmic rays while in space. He is also considered to be one of the smartest men alive.
The second most powerful member of the Fantastic Four and the brother of Sue Richards. Due to an accident caused by cosmic radiation in space, he can manipulate fire, turn his entire body into it and fly.
Ben Grimm - The Thing - is the original, quintessential tough-guy of the Marvel Universe but because of his transformation he's also the tragic member of the Fantastic Four. Ben was an ace test-pilot until exposure to intense cosmic radiation mutated him into a rock-skinned monster with immense superhuman strength. Ben's exterior is much harder than stone and he has a gruff disposition, but inside he has a heart of gold.
Sharon Ventura was a former pro-Wrestler who fell in love with the Thing and subsequently joined the Fantastic Four as Ms. Marvel. During one adventure, she was exposed to Cosmic rays which mutated her body into a rock-like form similar to the Thing's.
Cannonball is a mutant with the ability to propel himself through the air like a human rocket. Over the years, Sam has grown from a New Mutant, to the leader of the original X-Force, to a full-fledged X-Man. He currently teaches at the Jean Grey School and is an Avenger.
One of the founding X-Men, Dr. Hank McCoy is a mutant possessing animal-like strength and agility. Despite being covered in blue fur and resembling a ferocious beast he possesses an astounding intellect and superb wit. Beast is currently helping the Jean Grey School run, while also serving as an Avenger.
The first X-Man, Scott Summers possesses the mutant ability to emit powerful force blasts from his eyes. He is visually distinctive for the ruby quartz visor he wears to control his devastating power. A born leader, Cyclops succeeded his mentor Professor X to command the X-Men.
With the cerebral ability to understand all languages, Cypher was considered to be one of the weakest New Mutants. He was best friends with Warlock, but was killed while defending Wolfsbane in battle. He was forcibly resurrected during Necrosha, and soon returned to the X-Men as a powerful strategist.
Gifted with the power to control the forces and elements that govern weather Storm stands among the most powerful mutants on the planet. A former thief, "goddess", and queen, Storm is a veteran member and mainstay of the X-Men. She is the team's field leader and the Headmistress of the Jean Grey School.
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6 Fun Facts about Mice
Spotting a mouse scurrying through your home is many people's worst nightmare. Because mice are so common, however, the likelihood of an infestation happening in your home is quite high. However, mice are actually quite surprising little creatures. They are smart, social and actually very clean. While you probably still don't want them taking up… Read More


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Never Let Me Go
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Release:September 3, 2010
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Booker TBooker Tio Huffman, Jr.
- Birthdate: 03/01/1961 (age 54)
- Height: 6'3"
- Weight: 256 Ib
Booker is best known for his time in World Cham...
Read More »-- The Rhodes Brothers vs. The Real Americans for the WWE tag-team titles will take place on this week's WWE SmackDown broadcast. The show will be taped tomorrow night in Atlanta, GA. We'll have live **SPOILERS** of the show here on the website.
-- The local advertising for next Monday's RAW taping in Uniondale, Long Island at the Nassau Coliseum features Randy Orton vs. The Big Show and John Cena vs. Alberto Del Rio in Survivor Series rematches. The local advertising also lists "The McMahons, Triple H and more." Will Vince McMahon return next week?
-- Josh Mathews, Booker T, Mick Foley and Bret Hart will be the "expert panel" on Sunday's WWE Survivor Series kickoff show.


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Originally Posted by Octy
I knew you would pick Darren as your actor, Kamie. I knew it. XD
And that kid from Icarly?? Eh...he's not...Bradish enough. ^^;
I just realized. Didn't Bradish used to mean 'cums in two seconds'? XD
I thought it was taking 2 hours with it. Although I probably just misinterpreted it. :P
@Kamex: Man! I'm eating breakfast! But yeah, that's what it what it originally meant. What I meant by it in this thread....it doesn't have the 'Brad' going on, ya know? XD
@Enzap: No, Kamie is right. 2 seconds. Jake made it. XD
@Enzap: That's a lot of time. XD
Enzap, that's only with Matt. Luna was unable to make Brad last that long.
@Octy: You eat breakfast at noon?
Oh yeah... that's right. lol
I just realized something... *tries not to imagine Nathan Kress and Darren kissing*
@Kamex: So? It just proves Luna's better at sex. XDDDD
and I woke up late. I stayed up until 3, doing important work. I woke up at 11:30. ^^;;
@Buzz: No! Nathan Kress is not gonna be Brad. We need someone better. XD
1 - Luna's not better XD Trust me.
2 - I can't think of anyone to play Brad XD
1. Yes. She makes him climax a hell of a lot faster. XD
2. Scotty?


text:
Arizona Cardinals
Arizona Cardinals' Arians focused on fixing issues, getting ready for Lions
Still fresh off his team's loss to the St. Louis Rams, Arizona Cardinals head coach Bruce Arians now has to fix any issues his team had and prepare for their matchup with the Detroit Lions.
On The Dan Bickley Show with Vince Marotta on Arizona Sports 620 Monday, Arians talked about some of the things he's hoping to fix from the game as well as previewed some of the key matchups against the Lions.
"When we leave the building today at 5 o'clock, I want everything corrected from this game," Arians said. "That's our coaches' job, that's our players' job."
Aside from the offensive line troubles that have already been well documented, one area that needs fixing according to Arians is putting pressure on the quarterback.
"We had good inside pressure, it's the outside pass rushers that need to stick to their techniques and do what they do best," Arians said. "Don't try to create on the run someone you're not. We were a little bit out of sync on who we were in that ballgame."
When asked about the Lions, Arians first mentioned Detroit's imposing defensive line, consisting of rookie Ezekial Ansah, Nick Fairly and Ndamukong Suh, who received a hefty suspension from the NFL for a hit during his team's Week 1 win over Minnesota.
Arians also talked planning for running back Reggie Bush, who's coming off a great all-around performance in week one and is used in a multitude of ways by the Lions.
"They have those great wide receivers and you're going to double-team them a lot so you're going to have Reggie sitting there one-on-one and that's the type of running game they want," Arians explained.
Between Bush and All-Pro wide receiver Calvin Johnson, the Cardinals defense will have their hands full against the Lions, who defeated the Minnesota Vikings 34-24 on Sunday.
Most Popular
Cardinals Audio
- What on Your Mind? - Thursday July 2Vince Marotta vents about uniform numbers, airline fees, and Dwayne "the Rock" Johnson
- Top 5: Career Comebacks - Tuesday June 30Who had the best career comeback? Was it Jordan, Travolta, Warner, or someone else?
- Bickley Blast - Tuesday June 30Is the end near for Roger Goodell?
- Hardware - Monday June 29The Justin Bieber award, the You're Just Mean award, and more of the best/worst of the weekend
- John Clayton, ESPN NFL insider - Friday June 26John Clayton says Denver was never close to trading Peyton Manning.


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News >> Browse Articles >> Crime News
Marine veteran pulls gun to stop woman from being beaten
Source: WISN
March 15, 2013
News >> Browse Articles >> Crime News
Source: WISN
March 15, 2013
over 2 years ago
11372 Comments
"""Despite Blackmore's actions, Jeri Bonavia of the Wisconsin Anti Violence Effort said concealed carry is a bad idea."""
Are you kidding me? She could be dead otherwise!
States and politicians should wake up and support the 2nd Amendment and the need for Law Abiding Citizens to defend themselves and others in these violent times especially when police forces are shrinking and response times are increasing...
over 2 years ago
44584 Comments
Bump Max.
over 2 years ago
Mr. Blackmore was equipped and prepared to make a difference! Guns level the playing field,and can and do save lives!


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Ichiban Kuji One Piece Opening a New Era: Luffy
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Figures For Sale Part 1 - One Piece Stuff
I will have to get rid of quite a few figures from my collection. All are in great, like new condition. Every figure comes with a box unless otherwise mentioned. Figures are original - bought from reputable stores like AmiAmi, Mandarake or Yahoo Auctions. One piece stuff comes with toei gold sticker...
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NOS#741-19151NOS Emblem
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text: Bookmarks Hazardous materialsPrint-Friendly NavigatorHazardous materials are substances that could harm human health or the environment. Hazardous means dangerous, so these materials must be handled the right way.Hazard communication, or HazCom, is teaching people how to work with hazardous materials and waste.There are many different kinds of hazardous materials, including:Chemicals, like some that are used for cleaning Drugs, like chemotherapy to treat cancer Radioactive material that is used for x-rays or radiation treatments Human or animal tissue, blood, or other substances from the body that may carry harmful germs Gases that are used to make patients sleep during surgery Hazardous materials can harm you if they:Touch your skin Splash into your eyes Get into your airways or lungs when you breathe Cause fires or explosions Your hospital or workplace has policies about how to deal with these materials. You will receive special training if you work with these materials.I Would Like to Learn About:Expand All Collapse AllAlternate namesHazCom; Hazard communication; Material Safety Data Sheet; MSDSWatch out for hazardous materialsKnow where hazardous materials are used and stored. Some common areas are where:X-rays and other imaging tests are done Radiation treatments are performed Medicines are handled, prepared, or given to patients -- especially cancer treatment drugs Chemicals or supplies are delivered, packed for shipping, or thrown away Always treat any container that does not have a label like it is hazardous. Treat any spilled substance the same way.If you do not know if something you use or find is harmful, be sure to ask.Labels and signsLook for signs before you enter a patient's room, a lab or x-ray area, a storage closet, or any area you do not know well.You may see warning labels on boxes, containers, bottles, or tanks. Look for words like:Acid Alkali Carcinogenic Caution Corrosive Danger Explosive Flammable Irritant Radioactive Unstable Warning A label called the Material Safety Data Sheet (MSDS) will tell you if a material is hazardous. This label tells you:Names of the hazardous chemicals or substances in the container Facts about the substance, such as the odor or when it will boil or melt How it could harm you What your symptoms could be if you are exposed to the material How to safely handle the material and what protective equipment to wear when you handle it Protective equipmentPersonal protective equipment (PPE) is special equipment you wear to create a barrier between you and germs. This barrier reduces the chance of touc...Read Article Now Book Mark Article What steps to take before more skilled or trained professionals come to help If the material could cause a fire or explosion, and what to do if this happens What to do if a spill or leak occurs What to do if there is danger from the material mixing with other substances How to safely store the material, including what temperature to keep it at, if moisture is safe, and whether it should be in a room with good airflow Work safely If you find a spill, treat it like it is hazardous until you know what it is. This means:Put on personal protective equipment (PPE), such as a respirator or mask and gloves that will protect you from chemicals. Personal protective equipment (PPE)Personal protective equipment (PPE) is special equipment you wear to create a barrier between you and germs. This barrier reduces the chance of touc...Read Article Now Book Mark Article Use disinfectant wipes to clean up the spill and put the wipes in double plastic bags. Contact waste management to clean the area and to throw away the supplies you used to clean up the spill. Always treat any unlabeled container as if it contains hazardous materials.Put the container in a bag and take it to waste management to be thrown away. Do not pour the material down the drain. Do not put the material in the normal trash. Do not let it get into the air. If you work with hazardous materials:Read the Material Safety Data Sheet (MSDS) for all materials you use. Know what type of PPE to wear. Learn about exposure risks, such as whether the material can cause cancer. Know how to use the material and how to store it or throw it away when you are done. Other tips include:Never enter an area where radiation therapy is taking place. Always use the safest container to move materials from one area to another. Check bottles, containers, or tanks for leaks. Related InformationOpen ReferencesReferencesOccupational Safety and Health Administration. Healthcare. https://www.osha.gov/SLTC/healthcarefacilities/index.html. Accessed February 24, 2014.BACK TO TOP Review Date: 2/3/2014 Reviewed By: Jatin M. Vyas, MD, PhD, Assistant Professor in Medicine, Harvard Medical School; Assistant in Medicine, Division of Infectious Disease, Department of Medicine, Massachusetts General Hospital. Also reviewed by David Zieve, MD, MHA, Isla Ogilvie, PhD, and the A.D.A.M. Editorial team. The information provided herein should not be used during any medical emergency or for the diagnosis or treatment of any medical condition. A licensed medical professional should be consulted for diagnosis and treatment of any and all medical conditions. Call 911 for all medical emergencies. Links to other sites are provided for information only -- they do not constitute endorsements of those other sites. © 1997- A.D.A.M. content is best viewed in IE9 or above, Firefox and Google Chrome browser.Content is best viewed in IE9 or above, Firefox and Google Chrome browser.


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The Message audio Bible is now available on Bible Gateway! Click to learn more.
27:28 robe. Lit. "cloak," of a Roman soldier. Matthew calls the robe "scarlet," although Mark and John call it "purple." Matthew's detail emphasizes the indignity and may hint at the popular conception of the Messiah as a military and political deliverer.


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Wednesday, September 1, 2004
© Copyright 2015 Clayton News Daily
By Greg GelpiClayton County SAT scores rank well below the state average, which ranks second from the bottom in the country. The national average for the standardized test is 1,026. Georgia, which was at the bottom last year, scored an average of 987, inching just ahead of South Carolina's 986. Clayton County students averaged 901. "While we are pleased that our total scores reflect an increase for the first time in three years, we are fully aware that our overall performance is not where we want it to be," Clayton County schools Superintendent Barbara Pulliam stated in a press release. "We are including these SAT results in our study of student performance data with an ultimate goal of developing initiatives that will better prepare our students for academic success." The Clayton County school system currently offers two SAT study sessions during the summer. The school system produced an SAT study video and provides Kaplan SAT software. "By improving academic rigor, by increasing the number of advance placement courses, by ensuring that our students can read for comprehension, we are building a foundation that can lead to a better performance by our students on such tests as the SAT," Pulliam said. She commended all of the schools, especially Jonesboro High School, the county's only school to beat the state average. "SAT scores are very important because many colleges use this as part of the criteria for admissions," said Sid Chapman, the president of the Clayton County Education Association, adding "they don't always tell the whole story." Scores are lower in Clayton County due in part to the exodus of good teachers, Chapman said. "Without a doubt, we have lost many many excellent teachers to other systems in recent years," he said. "There are a lot of good teachers here now, but I always hear that they are talking about leaving. To maintain them, the teachers must be empowered." Classroom size must be addressed, Chapman said. High school classes are "jam packed" and the "teacher-student ratio has got to change," he said. "I think the students can perform," he said. "We just need to create an environment that is conducive to learning." Despite ranking second to last in the country, the State Department of Education points out that Georgia is improving faster than the nation. In the past 10 years, the state upped its verbal scores by 20 points as compared to the nation's 9-point increase and improved math scores by 19 points versus the nation's 14-point increase. "This is great news, but we recognize that there's still a lot work to do," State Superintendent of Education Kathy Cox said. "We are already moving ahead with our statewide SAT improvement efforts and I am optimistic that this is an upward trend we can continue." Since 1994, the state has improved or remained the same each year. The state experienced a 3-point increase this year, while the nation saw no increase. Clayton College & State University uses the University System minimum SAT requirements for admission. The minimum verbal score is 430 and minimum math score is 400. The Associated Press contributed to this story.
More like this story
- Pulliam focuses on future ( March 10, 2005 )
- Clayton schools post SAT improvements ( August 26, 2009 )
- Georgia SAT scores in the basement ( August 31, 2005 )
- Pulliam reflects on first year ( February 11, 2005 )
- Clayton's SAT scores decrease ( August 29, 2003 )


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The Smooth Fox Terrier, also known as the Fox Terrier, the Smooth-haired Fox Terrier, the Smooth-coated Fox Terrier and most affectionately as "the gentleman of the terrier world," is an old English breed that has been shown in the United States for more than a century. It developed in the 17th century as a hunting and sporting dog that specialized in flushing foxes and other vermin from their dens. Its keen senses of sight and smell, together with its size and stamina, made it especially well-suited to that task. The Smooth Fox Terrier is, as they say, "all terrier." It is plucky, active, short-backed and intently focused on whatever job it is given to do. It is used less to hunt fox and rodents now than it was historically and is more commonly used as a show dog and a companion. Potential owners should remember that this small terrier has an almost insatiable instinct to dig and is quite vocal. The Smooth Fox Terrier was recognized by the American Kennel Club as a breed distinct from the Wire Fox Terrier in 1995. The combined breed was admitted into the AKC, with a smooth and a wire variety, in 1885.
The mature male Smooth Fox Terrier should not exceed 15½ inches at the withers and should not be more than 12 inches from withers to the root of the tail in length, with the female being slightly smaller but in the same proportion. The adult male in show condition usually weighs about 18 pounds, with a bitch being about 16 pounds in the same condition. The breed's coat is smooth and flat but also hard, dense and abundant. White predominates with a few black patches, and brindle, red or liver markings are not preferred, because they too closely resemble the color of the fox. Its tail typically is docked to about ¾ of its original length and is set high and held upright.
The Fox Terrier has been shown in the United States as one breed with two varieties, the smooth and the wire, since it was first recognized by the American Kennel Club in 1885. In 1984, the AKC approved separate standards for the Smooth Fox Terrier and the Wire Fox Terrier, and breeds were recognized as fully distinct effective June 1, 1995. Experts believe that the two fox terriers developed very differently. The Wire Fox Terrier
Fox Terriers are spunky, fearless, loyal dogs who adore the outdoors as much as they enjoy people. They were developed to work as part of fox hunting parties, bolting foxes from their dens out into the open where Foxhounds and hunters then took over. Their small size allowed them to get into the foxes dens, and t heir long legs helped them keep up with the hounds. Today, Fox Terriers still enjoy running and digging,
The Smooth Fox Terrier is an active, lively dog with a short white coat and black or brown markings. They are small but strong and powerful with well-defined muscles - qualities necessary in the world of fox hunting. The skull is flat and tapering and the ears fold forward in a V. The eyes are small, deep set and dark in color. The head is wedge shaped and chiseled, the neck strong. The tail is
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Present address: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 36703 HA, Wageningen, The Netherlands.
AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis
Article first published online: 26 JUL 2007
DOI: 10.1111/j.1365-313X.2007.03211.x
The Plant Journal
Volume 52, Issue 1, pages 114–123, October 2007
Additional Information
How to Cite
Dharmasiri, N., Dharmasiri, S., Weijers, D., Karunarathna, N., Jurgens, G. and Estelle, M. (2007), AXL and AXR1 have redundant functions in RUB conjugation and growth and development in Arabidopsis. The Plant Journal, 52: 114–123. doi: 10.1111/j.1365-313X.2007.03211.x
Publication History
- Issue published online: 27 JUL 2007
- Article first published online: 26 JUL 2007
- Received 26 February 2007; revised 31 May 2007; accepted 4 June 2007.
- Abstract
- Article
- References
- Cited By
- auxin;
- embryogenesis;
- related to ubiquitin;
- Skp1;
- cullin;
- F-box protein;
- ubiquitin
Cullin-RING ubiquitin-protein ligases such as the Skp1, cullin, F-box protein (SCF) have been implicated in many growth and developmental processes in plants. Normal SCF function requires that the CUL1 subunit be post-translationally modified by related to ubiquitin (RUB), a protein related to ubiquitin. This process is mediated by two enzymes: the RUB-activating and RUB-conjugating enzymes. In Arabidopsis, the RUB-activating enzyme is a heterodimer consisting of AXR1 and ECR1. Mutations in the AXR1 gene result in a pleiotropic phenotype that includes resistance to the plant hormone auxin. Here we report that the AXL (AXR1-like) gene also functions in the RUB conjugation pathway. Overexpression of AXL in the axr1-3 background complements the axr1-3 phenotype. Biochemical analysis indicates that AXL overexpression restores CUL1 modification to the wild-type level, indicating that AXR1 and AXL have the same biochemical activity. Although the axl mutant resembles wild-type plants, the majority of axr1 axl-1 double mutants are embryo or seedling lethal. Furthermore, the axl-1 mutation reveals novel RUB-dependent processes in embryo development. We conclude that AXR1 and AXL function redundantly in the RUB conjugating pathway.
The phytohormone auxin (indole-3-acetic acid or IAA) is an essential regulator of plant growth and development from embryogenesis to senescence. To understand how plants respond to auxin at the cellular and molecular level, several mutant screens have been conducted in the model plant Arabidopsis, and a number of auxin-resistant mutants have been identified. One of these mutants, auxin resistant1 (axr1) (Lincoln et al., 1990), has a pleiotropic phenotype that includes defects in root gravitropism, lateral root formation, root hair growth, apical dominance, plant height and fertility. Molecular and biochemical studies indicate that AXR1 encodes a subunit of a heterodimeric RUB (related to ubiquitin, Nedd8 in animals)-activating enzyme (Leyser et al., 1993; del Pozo et al., 1998). The other subunit in this enzyme is called ECR1 (del Pozo et al., 1998).
The ubiquitin–proteasome pathway consists of three proteins or protein complexes called the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligase (E3). The E3s are responsible for the specificity of the pathway, and constitute very large families in plants and other eukaryotes (Pickart, 2001). One superclass of E3s are the multisubunit cullin-RING ligases (also called CRLs), each of which contains a cullin protein, a RING protein and a substrate receptor protein (Petroski and Deshaies, 2005). The best-known CRLs are the SCF (Skp1, cullin, F-box protein) E3s, in which the F-box protein functions as the substrate receptor. SCFs are found in all eukaryotes and are responsible for the degradation of a wide variety of proteins involved in many different cellular processes (Petroski and Deshaies, 2005). In Arabidopsis, SCFs have been implicated in senescence (Woo et al., 2001), apical dominance (Stirnberg et al., 2002), circadian rhythm (Nelson et al., 2000; Somers et al., 2000), flower and meristem development (Samach et al., 1999; Zhao et al., 1999) (Ingram et al., 1997), phytochrome A signaling (Dieterle et al., 2001), self-incompatibility (McClure, 2004) and phytohormone signaling (Gray et al., 1999; McGinnis et al., 2003; Ruegger et al., 1998; Sasaki et al., 2003; Xie et al., 1998). The best-characterized SCF in plants is SCFTIR1, which is implicated in auxin signaling. SCFTIR1 and the related E3s SCFAFB1–SCFAFB3 promote the degradation of the Aux/IAA transcriptional repressors in an auxin-dependent manner (Dharmasiri et al., 2003a;Gray et al., 1999; Gray et al., 2001). Recent studies have shown that auxin binds directly to TIR1 to promote interaction between the SCF and the Aux/IAA substrate (Dharmasiri et al., 2005a; Kepinski and Leyser, 2005).
Like ubiquitin, RUB is conjugated to other proteins by E1 and E2 enzymes. In Arabidopsis, these proteins are the AXR1-ECR1 heterodimer and RCE1, respectively (del Pozo et al., 1998, 2002). Studies in Arabidopsis as well as many other species indicate that the function of the RUB conjugation pathway is to attach RUB to members of the cullin family of proteins, including the CUL1 subunit of the SCF. In the case of the SCF, RUB modification of CUL1 is required for normal E3 function (Petroski and Deshaies, 2005). Thus, mutations in components of the RUB conjugation pathway, such as axr1 and rce1, result in stabilization of the Aux/IAA proteins and decreased auxin response (Dharmasiri et al., 2003b; del Pozo et al., 1998). Furthermore, the double mutant axr1 rce1 is severely affected in RUB modification of CUL1, and displays an early seedling lethal phenotype (Dharmasiri et al., 2003b). This phenotype is similar to that of quadruple mutants lacking the TIR1/AFB auxin receptors, as well as bdl/iaa12 and mp/arf5 mutants, suggesting that RUB modification of CUL1 is essential for the degradation of SCFTIR1 targets and normal embryogenesis (Dharmasiri et al., 2003b; Weijers and Jurgens, 2005). Plants that are deficient in the related RUB1 and RUB2 proteins are arrested early in embryogenesis, confirming the importance of RUB conjugation to plant growth and development (Bostick et al., 2004). The axr1, rce1 and rub mutants are also affected in other hormone processes, including the jasmonic acid (JA) response and the regulation of ethylene biosynthesis, underscoring the general role of RUB conjugation in SCF function (Dharmasiri et al., 2003b; Larsen and Cancel, 2004).
Although the RUB conjugation pathway is clearly important for plant growth and development, null axr1 alleles are viable (del Pozo et al., 1998). A search of the Arabidopsis genome identified AXL (AXR1-like), which encodes a protein with a high level of similarity to AXR1. Here we report on the characterization of AXL. Our studies show that AXL has a similar function to AXR1 in the RUB conjugation of CUL1. Genetic studies indicate that AXL and AXR1 are functionally redundant. Thus, the loss of both genes results in severe affects on plant growth and development, confirming that the RUB conjugation pathway is essential for viability.
The AXL gene can replace AXR1
The relatively weak phenotype of the axr1 mutants, compared with the embryo lethal phenotype of mutants that are deficient in the RUB1 and RUB2 genes, prompted us to search for a second gene that can provide RUB-activating activity (Bostick et al., 2004). The Arabidopsis genome contains a single gene (At2 g32410) with strong similarity to AXR1, which we named AXL (AXR1-LIKE). The protein encoded by this gene is 80% identical to AXR1. Based on this similarity, we hypothesized that AXL may also be part of a RUB E1 enzyme. To investigate this, we generated a construct in which the AXL cDNA was placed adjacent to the CaMV35S promoter, and introduced this construct into axr1-3 plants. Four transgenic lines were selected for further characterization. In all four lines, the wrinkled rosette leaf phenotype characteristic of axr1-3 plants was restored to a wild-type appearance (Figure 1a). In addition, the 35S:AXL transgene rescued other aspects of the axr1-3 phenotype, including inflorescence height and fertility (data not shown). Moreover, the transgene restored normal auxin sensitivity to the roots of axr1-3 seedlings (Figure 1b). Consistent with these results, each line had a high level of AXL RNA and AXL protein (Figure 1c).
Previous studies have shown that the level of RUB-CUL1 is reduced in the axr1 mutants (del Pozo et al., 1998). Thus, we investigated whether overexpression of AXL leads to increased levels of modified CUL1. As expected, RUB modification of CUL1 is decreased in axr1-3 plants as a result of reduced RUB E1 activity (Figure 1c). However, the level of RUB-CUL1 was restored in each transgenic line relative to wild-type levels, indicating that AXL can replace AXR1 in the RUB conjugation pathway.
AXL gene expression
To investigate the pattern of AXL expression we performed a reverse transcription polymerase chain reaction (RT-PCR) experiment using RNA isolated from various tissues (Figure 2a). The abundance of AXL mRNA was extremely low compared with AXR1 mRNA (data not shown). Although AXL RNA was detected in all the tissue types examined, root tissues showed the highest RNA abundance. However, at least 50 PCR cycles were required to obtain a band detectable by standard agarose gel electrophoresis. The relatively low level of expression of AXL in all tissues is also confirmed by microarray analysis (Schmid et al., 2005) (http://www.weigelworld.org/resources/microarray/AtGenExpress). Notably, in these studies, the expression patterns of AXR1 and AXL are very similar, but AXR1 is consistently expressed at a level that is higher than AXL expression, by about one order of magnitude. Furthermore, a query of available microarray data using Genevestigator shows that AXL is expressed at a low level under all conditions examined (Zimmermann et al., 2005).
Isolation of a T-DNA insertion line in the AXL gene
To investigate the function of AXL, we screened a T-DNA insertion mutant population in the Wassilewskija (Ws) ecotype, and identified a line carrying a single T-DNA insertion in AXL, designated axl-1. Like AXR1, the AXL gene consists of 14 exons and 13 introns. The exon/intron boundaries in the two genes are conserved, indicating that the two genes were produced by a relatively recent duplication event. The T-DNA insertion is located in the 6th intron (Figure 2a). RNA blot analysis indicated that axl-1 is lacking full-length AXL mRNA (Figure 2c).
Despite the fact that axl-1 appears to be a null allele, homozygous axl-1 plants do not exhibit an obvious mutant phenotype. The rosettes are normal, and mutant plants bolt at the same time as the wild-type plants, producing a normal and fully fertile inflorescence. In contrast, even the moderately auxin-resistant axr1-3 mutant displays abnormal floral features such as short stamens, reduced fertility and short siliques (Lincoln et al., 1990). However it is important to note that axl-1 plants may exhibit a phenotype under conditions that we have not examined, possibly reflecting a novel function of the AXL protein.
The axl-1 and axr1-12 mutations have synergistic effects on plant growth and development
To determine if AXL might function redundantly with AXR1, we generated a double mutant line between axl-1 and axr1-12, a strong AXR1 allele. The resulting F1 plants were similar to wild type in appearance (data not shown). The segregating F2 population was studied in detail to investigate the effect of AXR1 and AXL gene dose on plant morphology. The AXR1/axr1-12, axl-1/axl-1 plants were similar to wild type with respect to overall morphology and fertility (data not shown). However, as shown in Figure 3a, homozygous axr1-12 plants that are heterozygous for axl-1 display severe growth defects. These seedlings developed short, slow growing roots compared with wild-type roots, and were arrested at the late seedling stage.
In addition, plants that were homozygous for both mutations displayed severe growth and developmental defects. The phenotype of these plants resembled the bdl/iaa12 and mp/arf5 mutants (Berleth and Jurgens, 1993; Hamann et al., 1999) (Figure 3a). These seedlings did not segregate at the expected 1/16 ratio. Instead, we identified three double mutant seedlings in 176 F2 plants, suggesting the homozygous double mutants are arrested during embryogenesis. A further characterization of embryo defects in the axr1 axl-1 double mutant is described below.
Auxin has been implicated in the patterning of the root meristem, and previous studies have shown that the axr1 mutants display a reduction in the number of columella cells (Sabatini et al., 1999). Similarly, the loss of the auxin receptors TIR1 and AFB1, AFB2 and AFB3, has a pronounced effect on the development and maintenance of the root meristem (Dharmasiri et al., 2005b). To determine the contribution of AXL to the root meristem, we stained wild-type, axl-1, axr1-12 and AXL/axl-1 axr1-12/axr1-12 seedling roots with Lugol solution and examined root meristem organization. The axl-1 single mutant did not display a defect in meristem organization (data not shown). As described previously, we frequently observed a slight decrease in the number of columella columns in axr1-12 seedlings compared with wild type (Figure 3b) (Sabatini et al., 1999). In contrast, the removal of a single AXL copy resulted in dramatic changes in the columella. The columella of AXL/axl-1 axr1-12/axr1-12 root tips is highly disorganized during early seedling root growth, and is completely disrupted at the time when root growth ceases (Figure 3b).
Auxin is also known to play a role in vascular patterning in plants (Berleth et al., 2000), and loss of AXR1 and RCE1 activities results in defects in vascular development (Dharmasiri et al., 2003b). To investigate the impact of loss of AXL on vascular patterning, we examined the cotyledons of axr1-12 axl-1 double mutants. As predicted, axr1-12 axl-1 double mutants showed greatly reduced vascular development similar to that in mp, bdl, axr6 and the axr1-12 rce1 double mutant (Dharmasiri et al., 2003a; Hamann et al., 1999; Hellmann et al., 2003; Przemeck et al., 1996) (Figure 3c).
In summary, although not apparent in the axl-1 single mutant, the axr1 axl-1 double mutant reveals an important function for AXL throughout plant development that is redundant with the function of AXR1.
RUB modification of CUL1 is affected in axr1 axl-1 double mutants
To investigate the relationship between seedling lethality and RUB modification of CUL1, the level of RUB-CUL1 was examined in wild type and in various mutant genotypes. As shown in previous studies, the axr1-12 mutant exhibits reduced RUB modification of CUL1 (Figure 3d) (Dharmasiri et al., 2003a). However, the axl-1 mutant has a normal level of RUB-CUL1 that is in keeping with the lack of any growth defect in these plants. Plants with the genotype AXL/axl-1 axr1-12/axr1-12 had a slight but consistent reduction in RUB-CUL1 levels (Figure 3d), which is consistent with the severe phenotype displayed by these seedlings. However, the relatively rare homozygous double mutant seedlings are completely deficient in RUB-modified CUL1 (Figure 3d).
Auxin response is reduced in axr1-12 axl-1 double mutants
In order to study the role of AXL on auxin response, we determined the effects of auxin on root growth in single and double mutants. As expected, the axr1-12 mutant was clearly resistant to auxin (Figure 4a). In contrast, the axl-1 mutant was very similar to the Ws parental line. However, the axr1-12/axr1-12 AXL/axl-1 line was nearly completely resistant to auxin, a much more severe phenotype than for axr1-12 alone. This result demonstrates that AXL functions in auxin response.
Previous studies have shown that AXR1 is also required for the JA response (Tiryaki and Staswick, 2002). This requirement is probably related to SCFCOI1, an E3 known to be required for JA signaling (Xie et al., 1998). To determine if AXL is also required for the JA response, we performed root growth assays with our mutant lines (Figure 4b). As for auxin, we found one axl-1 gene dramatically reduces the JA response in axr1-12 plants.
To obtain additional evidence for a role for AXL in auxin signaling, we also examined expression of the auxin-regulated genes IAA2, IAA5 and IAA7. The induction of IAA2 and IAA5 was dramatically reduced in axr1-12/axr1-12 AXL/axl-1 plants compared with the wild type (Figure 4c), and was also reduced compared with axr-12. Induction of IAA7 was also reduced, although to lesser extent.
Previous studies show that defects in SCFTIR1 and/or SCFAFBs stabilize the Aux/IAA proteins in Arabidopsis (Gray et al., 2001; Dharmasiri et al., 2005b). To determine whether Aux/IAA degradation in response to auxin treatment is altered in the axl-1 mutants we crossed the HS::AXR3NT-GUS transgene into axl-1 and AXL/axl-1 AXR1/axr1-12 lines and screened for homozygous GUS axl-1 axr1-12 plants. The resulting seedlings were heat shocked for 2 h at 37°C to induce GUS expression, and AXR3-GUS stability in the presence of auxin was tested using GUS histochemical assays. As shown in Figure 4d AXR3-GUS is degraded in wild type, but is relatively stable in axr1-12 plants. In axl-1 axr1-12 double mutants GUS staining is even stronger than for axr1-12, suggesting a strong correlation between Aux/IAA stability and the severe growth and developmental defects of double mutant plants.
The RUB conjugation pathway is required during early embryogenesis
The AXR1 protein accumulates throughout plant development, starting very early during embryogenesis in the zygote (del Pozo et al., 2002). However, it is not known whether its function is required at this early stage. Because AXR1 appears to be the major RUB E1 (see above), we first investigated embryo development in the null allele axr1-12, and found no deviation from the wild type (data not shown). As AXR1 shares a number of redundant functions with AXL, we next examined embryo development in the axl-1 single and axr1-12 axl-1 double mutants. No embryo defects were found in axl-1 (n = 92). In contrast, very strong embryo defects were observed in homozygous axl-1 plants segregating the axr1-12 mutation. These phenotypes fell into two classes based on the severity of their defects. Embryos in one class (7% of total embryos; n = 116) were very severely affected and displayed developmental arrest after a few rounds of, often abnormal, cell divisions. Examples of such embryos are shown in Figure 5, and show aberrant planes of cell division (Figure 5e), bloating of cells (Figure 5f) and occasionally failure to complete cell walls (Figure 5g).
A larger fraction of double mutant embryos (19% of total embryos; n = 116) were less severely affected, with patterning defects reminiscent of bdl, mp and tir1 afb2 afb3 mutants (Dharmasiri et al., 2005b) (Figure 5b,d). As the percentage of abnormal embryos is close to 25%, the double mutants appear to have a fully penetrant phenotype, without clear evidence for gametophytic defects. It is unclear what causes the variability in phenotype. As the rootless double mutant seedlings described above are found at a rate of 1–2%, it is likely that most of the double mutant embryos in the weaker class are either arrested late in embryo development or do not germinate. Nonetheless, AXR1 and AXL share redundant functions in early embryogenesis, and their activities are required for proper cell division and embryo patterning.
In addition to ubiquitin, eukaryotic organisms contain several ubiquitin-like proteins (Ubls) that are post-translationally conjugated to a variety of other proteins (Kerscher et al., 2006). Ubls have been implicated in diverse cellular processes including transcription, cell cycle control and autophagy (Kerscher et al., 2006). In the case of RUB/Nedd8, attachment of the Ubl to the cullin subunit of CRLs is required for CRL activation. The best-characterized CRL, the SCF complex, is involved in a wide variety of hormonal and developmental pathways in Arabidopsis (Moon et al., 2004). As in other organisms, modification of the CUL1 subunit of the SCF by RUB is necessary for normal function of the E3 complex. In this report we show that the AXL protein functions in RUB activation, and that the RUB conjugation pathway is essential for viability in Arabidopsis.
The AXL protein is part of a RUB E1
RUB is covalently attached to the cullin through the action of a heterodimeric activating enzyme and a RUB-specific E2 enzyme. In Arabidopsis, these enzymes are called AXR1-ECR1 and RCE1 respectively. Both the axr1 and rce1 mutants display severe growth defects as well as reduced auxin sensitivity (Dharmasiri et al., 2003b; del Pozo et al., 2002). However, both mutants produce some RUB-CUL1, suggesting that they are not completely deficient in RUB-activating and -conjugating activity, respectively. Consistent with this, the axr1-12 rce1 double mutant is more severe than either single mutant, with respect to both RUB-CUL1 levels and development. The AXL gene produces a protein that is 80% identical to AXR1, suggesting that it may also function in a RUB E1 enzyme. Indeed, overexpression of AXL in the axr1-3 mutant increases the level of RUB-CUL1 to wild-type levels, and restores the wild-type phenotype to mutant plants, strongly suggesting that AXL also functions in a heterodimeric RUB-activating enzyme. As there is only a single ECR1 gene in Arabidopsis, it is likely that AXL interacts with ECR1 to form this enzyme. Interestingly, the Arabidopsis genome also has two closely related genes that encode RUB-conjugating enzymes. One of these, called RCE1, is expressed at a relatively high level, and has an important role in RUB conjugation (Dharmasiri et al., 2003b). The second, RCE2, has not been characterized, but like AXL is expressed at a low level throughout development (Schmid et al., 2005).
The RUB-activating enzyme is essential for viability in Arabidopsis
Genetic studies in a wide variety of eukaryotes indicate that the RUB/Nedd8 pathway is essential for viability (Kerscher et al., 2006). The one exception is the yeast Saccharomyces cerevisiae, in which defects in the pathway have little or no effect on cell growth. In Arabidopsis, single mutants in AXR1 and RCE1 have severe defects, but are nonetheless viable (Dharmasiri et al., 2003b). However, plants that are homozygous for mutations in both genes die as young seedlings. Similarly, plants with mutations in two of the three RUB genes (RUB1 and RUB2) are embryo lethal (Bostick et al., 2004). Our results with the axl-1 mutant confirm that RUB conjugation is required for normal embryogenesis. The axr1-12 axl-1 mutant also exhibits an embryo or early seedling lethal phenotype, and appears to completely lack RUB-CUL1.
The axl-1 mutation reveals novel RUB-dependent processes in embryo development
Interestingly, the early embryo arrest of the axr1-12 axl-1 double mutant allows the definition of additional developmental events during which RUB-dependent CRL activity is required. So far, a series of mutant genotypes has been reported that progressively affect CUL1 levels or the RUB modification of CUL1. Intriguingly, all mutants analyzed so far are arrested either at or directly after the first cell division, or progress normally to the globular stage and fail to initiate a root. The cul1 knock-out mutant is zygote lethal (Shen et al., 2002), and weaker alleles either cause rootless seedlings or pleiotropic post-embryonic defects (Hellmann et al., 2003; Moon et al., 2007; Quint et al., 2005). Likewise, rub1 rub2 mutants are arrested at the two-cell stage of embryogenesis, and RNA knock-down causes pleiotropic post-embryonic defects (Bostick et al., 2004). In the case of the RUB conjugation pathway, the earliest defects reported so far are in axr1rce1 mutants (Dharmasiri et al., 2003b). These double mutant seedlings lack a root and, when examined microscopically, the earliest defect observed was the stage when the root is initiated (22% of 51 embryos in homozygous axr1-12 plants segregating rce1-1) (data not shown). Hence, it is unclear if RUB-dependent CRL activity is required after the first cell division and before root initiation. In addition, AXR1, RCE1 and CUL1 are all expressed broadly in early embryos at stages where no mutant phenotypes have been reported (Dharmasiri et al., 2003b; del Pozo et al., 1998, 2002; Shen et al., 2002). These results suggest that there are two checkpoints in early embryogenesis where CRL activity is critical for progression. Alternatively, it is possible that the full spectrum of RUB-dependent CRL activity has not yet been revealed, perhaps because of functional redundancy. Indeed, the severe axr1 axl-1 double mutant phenotype displays defects in stages ranging from the zygote to the dermatogen stage. These defects include aberrant cell division planes, abnormal swelling of cells and incomplete cell divisions. Although some effects may be indirect consequences of defects at earlier stages, these results show that many processes during early development require RUB modification of CULLINs, and that RUB modification is required throughout plant embryogenesis.
Experimental procedures
Plant material, growth conditions and treatments
The axl mutant is in the Ws ecotype, whereas axr1-3 and axr1-12 mutants are in the Columbia (Col-0) ecotype. The HS::AXR3NT-GUS has been described previously by Gray et al. (2001). Surface-sterilized Arabidopsis seeds were germinated on Arabidopsis thaliana salts (ATS) medium [1% sucrose, 5 mm KNO3, 2.5 mm KH2PO4 (pH 5.6), 2 mm MgSO4, 2 mm Ca(NO3)2, 50 μm CuSO4, 1 μm ZnSO4, 0.2 mm NaMoO4, 10 μm NaCl, and 0.01 μM CoCl2) with 0.8% agar. Plates were placed vertically in a growth chamber at 22°C under continuous light. Where necessary, 8–10-day-old seedlings were transferred to soil and grown at 22°C under continuous light.
For root growth assays, 5-day-old seedlings were transferred to ATS medium with or without hormones, and were grown vertically in a growth chamber at 22°C under continuous light for the designated times. To study auxin-induced gene expression, seedlings were grown on ATS for 6 days, transferred to liquid ATS medium and grown for indicated times with or without auxin. Seedlings were harvested at the end of treatments and used for RNA extraction.
Generation of transgenic lines
The AXL cDNA was cloned into pCR 2.1 vector and the correct sequence was confirmed by DNA sequencing. To construct the AXL overexpression lines, AXL cDNA was placed behind the CaMV 35S promoter in the binary vector pROKII and the plasmid construct was introduced into A. tumefecians strain GV3101. Arabidopsis mutant line axr1-3 was transformed by using the floral-dip method to generate the axr1-3(AXL) lines.
Isolation of T-DNA insertion line and generation of double mutants
The T-DNA insertion mutant line for the AXL gene (axl-1) was isolated by screening the University of Wisconsin mutant lines (Ws) using the gene-specific primer AXL1F (5′-CCACAAACTTGCAACCAGTTGAA-3′) together with the T-DNA left border primer JL202 (5′-CATTTTATAATAACGCTGCGGACATCTAC-3′). After selecting the mutant line, the T-DNA insertion site was confirmed by sequencing the PCR product using the JL202 primer.
To generate axl-1 axr1-12 double mutants, homozygous axl-1 plants were crossed with axr1-12 lines. As the double mutant is seedling lethal, resulting F2 progeny were genotyped by PCR to isolate lines that are homozygous for the axl-1 mutation and heterozygous for the axr1-12 mutation. Progeny from these plants were used for the experiments to characterize the phenotype of various genotypes. The HS::AXR3NT-GUS line was introduced to the parent heterozygous mutant line by crossing.
Northern, RT-PCR and protein blot analysis
To determine the expression of the AXL gene, total RNA was isolated from 100 mg of tissue collected from 10–12-day-old Arabidopsis seedlings using Tri-Reagent (Sigma, http://www.sigmaaldrich.com). Total RNA (10 μg) was separated on formaldehyde-agarose gel and transferred to nylon membrane. The blot was hybridized to the 32P-labeled AXL cDNA probe. Hybridization and washing of the blot was performed according to standard conditions and exposed to X-ray film. To analyze the expression of AXL in different tissues or developmental stages, total RNA was isolated from tissues using the Tri-Reagent. cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen, http://www.invitrogen.com) and oligo dT primer. The reverse transcription product (1 μl) was used as the template DNA to amplify the AXL sequence using specific primers running 50 cycles of PCR.
The expression of Aux/IAA genes in mutant plants was determined using 6-day-old seedlings treated with or without 20 μm 2,4-d for 1 h. Total RNA was extracted, separated and blotted as describe above. Blots were hybridized to probes prepared from cDNAs of IAA2, IAA5 or IAA7.
To analyze AXL protein abundance and the status of the CUL1 protein in various genetic backgrounds, total protein was isolated from 10–12-day-old seedlings using a denaturing buffer containing 125 mm Tris (pH 8.8), 1% sodium dodecyl sulphate (SDS), 10% glycerol and 50 mm Na2S2O3. Total protein was estimated by the Bradford method. Total protein (20 μg) was separated by SDS-polyacrylamide-gel electrophoresis and transferred onto polyvinylidene difluoride membrane and incubated with affinity-purified either anti-AXR1 or anti-CUL1 antibody. Protein bands were visualized by horseradish peroxidase conjugated rabbit IgG using enhanced chemiluminescence (ECL; Pierce, http://www.piercenet.com).
GUS staining and analysis
HS::AXR3NT-GUS seedlings (6 days old) were heat shocked at 37°C for 2 h in liquid ATS medium. The seedlings were collected by filtration and transferred into new medium. Samples were collected immediately (0 min) and after 60 min to stain for GUS activity (Jefferson et al., 1987).
Analysis of root tip morphology and cotyledon vascular patterns
Wild-type and mutant seedlings grown on vertical ATS media were stained with Lugol solution (Sigma) for 5 min for root morphology analysis. The seedlings were mounted on Hoyer's medium and observed under the microscope (Meinke et al., 1994). To examine cotyledon vascular patterns, seedlings were first fixed in ethanol:acetic acid:water (6:3:1) by vacuum infiltration and mounted on Hoyer's solution for visualization under dark field optics.
For embryo phenotypic analysis, axr1-12, axl-1 and plants segregating axr1-12 and axl-1 or axr1-12 and rce-1, were grown, and (in the case of axr1-12) pre-selected for the homozygous axr1-12 phenotype. Developing seeds were collected from several siliques of 5–10 independent plants, and directly mounted in a 8:3:1 (w:v:v) mixture of chloral hydrate, water and glycerol. Embryos were observed with a Nikon microscope (Nikon, http://www.nikon.com) equipped with Differential Interference Contrast optics and a Zeiss Axiocam camera (Zeiss, http://www.zeiss.com). Digital photos were recorded using Zeis axiovision software and processed using Adobe photoshop CS2 (Adobe, http://www.adobe.com).
Work in the author's lab was supported by grants from the NIH (GM-43644) and NSF (MCB-0519970) to ME.
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Accession number: NM_001036388 (AXL).


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2007 BEST Restaurant, General Excellence, North
Food lovers are advised to use the buddy system at Evenfall: You'll definitely need someone to shore up your self-control in the face of chef Scott Pelletier's seductive creations, which include pan-seared duck breast with lemon pappardelle, fava beans, and wild mushrooms, and orange-infused pound cake with mint macerated strawberries and strawberry sorbet. Besides, you'll want someone to ooh and aah along with you over the artistic presentation of your meal—and possibly drive you home after that last irresistible cocktail from Evenfall's master mixologists.
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Mitchell argued that trains are a "money-losing scheme" in the long run for states because they have to pay for maintenance and upkeep themselves once the federal dollars stop flowing. Supporters of high-speed rail argue that applies for all public infrastructure, not just trains.
The bulk of the money for the project is being sent to the coasts, namely California and New York. Governors in both states have expressed their willingness to work closely with the federal government to develop infrastructure and advance the train system.
The first part of the project includes upgrading railways like the one that carries travelers between Washington and Boston on America's only high-speed train, Amtrak's Acela. Next up is creating new high-speed corridors in places like California, the Pacific Northwest and the Southeast.
Insiders say the project got off to a rocky start. Despite the hefty investment from the federal government, the announcement early last year came at a time states were not quite ready to take projects off the ground.
But proponents of the plan say the stimulus jumpstarted states into moving quickly from the planning stage to the implementation stage.
"The stimulus came at a very critical time. The transit systems around the country have suffered from the economic downturn," Guzzetti said. "The stimulus was a real lifesaver for many of them."
Another challenge is that the Transportation Department hasn't previously implemented the large-scale, high-speed passenger rail program called for in the stimulus and lacks the experience necessary in overseeing its design and construction, as the agency acknowledged in a report last year.


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Carlisle man charged with eight indecent assaults on boy
Last updated at 11:17, Tuesday, 05 February 2013
A 38-year-old man accused of a string of sex assaults on a boy more than 20 years ago has appeared before a judge at Carlisle Crown Court.
Robert Cook, of Eldon Drive, Harraby, Carlisle, is accused of eight indecent assaults on the boy on dates between 1988 and 1990.
Six of them are alleged to have taken place between October 9, 1988 and October 11, 1990, and the other two between October 10, 1988 and October 11, 1990.
Cook was not asked to enter pleas to the charges but he had previously denied all of them when he appeared before local magistrates.
His barrister Kim Whittlestone told the judge his pleas would be "a firm not guilty".
Cook is expected to go on trial in July.
First published at 11:15, Tuesday, 05 February 2013
Published by http://www.newsandstar.co.uk


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Photo by Dana Gallagher
Leave a Review
The elements of this recipe are sound: nutmeg, pepper and salt for flavor, heavy cream for that decadent base, and nutty gruyere cheese for that wonderful gratin. But depending on the size of your sweet potatoes, the recipe will produce way too many sweet potato slices. I use a large sweet potato and one medium yukon gold for good flavor balance. I bake them in individual ramekins. This is not a recipe "to follow exactly."
It is rare when I make an Epicurious recipe that falls flat¿I mean never, until this. It was bland and not worth the effort. I followed the recipe exactly as written and what came out was just sliced up sweet potatoes in heavy cream
These potatoes have become a stable at my dinner parties. I add a bit more heavy cream and gruyere for a more cream consistency and top them with freshly grated parmesan cheese. There are never any left!
I actually followed the recipe and it was great! I don't think this would be good with cinnamon, onions, and cheddar though. It made just over 12 and making these in a muffin tin was a nice presentation and added to its charm.
Good recipe. We coarse grated the potatoes and added onions. Used cinnamon instead of nutmeg. I would just bake them in a casserole dish rather than the cupcake pan next time. A lot of work and made way more than 12. Also used grated sharp cheddar since we had to use what we had in the pantry! Will make it again.


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Hey there, I'm James Pizzurro, probably known to you as 'Grey Echo' or 'madkowa', if at all. I specialized in programming for the Source engine under the now defunct Agent Red Productions, but I did some compiling, mapping and animating work from time to time too. I was also an occasional guest host for Podcast 17.
No blog entries have been posted here. Boring yes, but as confucius says - if you have nothing good to say, why say anything at all? Check back later, perhaps something exciting will pop-up and be worth blogging about, like season 3 of the teletubbies.
Speak your mind, rant about games, mods and the development scene, it's simple and who knows maybe someone will read it and agree. It will feel right!


text: Home > Parenting > Child > How to raise a baby How to raise a baby All sections in Parenting » Child » Pregnancy Surprises help babies learn Unpredictable situations seem to make infants more interested in exploring and learning, a new study says. TALK Parenting forum » ASK The Paediatrician » Follow Health24 on Facebook » Quiz Are you ready for a baby? » How to stimulate your baby Baby born as truck kills mother US mom says her baby born without a nose is 'perfect the way he is' US mom from Alabama, whose baby was born with a rare condition known as complete congenital arhinia that in his case means he was born without a nose, has said that he is just 'perfect the way he is'. When a baby is unusually quiet There are many reasons why a baby could be crying. But what does it mean if your baby is unusually quiet, and when should you worry? The health benefits of breastfeeding Breastfeeding has many great health benefits for both baby and mother. Here's why most moms should try to breastfeed for at least six months or longer. Your complete guide to breastfeeding After the delivery of your baby, a breastfeeding consultant, your midwife or a maternity ward nurse will guide you slowly through the steps of breastfeeding. Breastfeeding in public Should or shouldn't you breastfeed in public? If you do, here are some handy hints to make it easier for you and those around you. " Latest comment on Health24 Rod Van Deventer says... Pheeeew What your poo tells you " What makes a mom breastfeed? The health benefits of breastfeeding are well-known and yet there are many other factors that influence a mother's decision on whether to breastfeed or not. Dealing with your new baby We take a look at weight gain, neonatal jaundice, your baby's first stool, visitors and much more. Why breast is best We've all heard that "breast is best". But why is breastfeeding so important? load more articles advertisement From our sponsors Soothe your family's coughs and colds Why hydration is important while exercising Taking charge of your health Man, take care of your health! Live healthier Up for grabs » Win with NuSeed Win with MySmartkid Win a Canderel hamper Win a LED 3D smart TV Stand a chance of winning a Samsung UA55H8000 Smart 55 inch Curved LED TV valued at R20 000 from Save Hyper! Medical marijuana » 'I used cannabis oil to cure my cancer' Could your doctor could be high? Dagga in medicine? Medicinal dagga: the dangers can't be ignored There is not sufficient evidence to support the potential medicinal benefit from cannabis use cautions an academic from Stellenbosch University.


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12 CFR 225.177 - Definitions.
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(1) Except as otherwise expressly provided, the term "financial holding company" as used in this subpart means the financial holding company and all of its subsidiaries, including a private equity fund or other fund controlled by the financial holding company.
(2) Except as otherwise expressly provided, the term "financial holding company" does not include a depository institution or subsidiary of a depository institution or any portfolio company controlled directly or indirectly by the financial holding company.
(b) What do references to a depository institution include? For purposes of this subpart, the term "depository institution" includes a U.S. branch or agency of a foreign bank.
(1) That is engaged in any activity not authorized for the financial holding company undersection 4 of the Bank Holding Company Act (12 U.S.C. 1843); and
(2) Any shares, assets or ownership interests of which are held, owned or controlled directly or indirectly by the financial holding company pursuant to this subpart, including through a private equity fund that the financial holding company controls.
(1) An executive officer of a company is any person who participates or has the authority to participate (other than in the capacity as a director) in major policymaking functions of the company, whether or not the officer has an official title, the title designates the officer as an assistant, or the officer serves without salary or other compensation.
(i) Any person, including a person with an official title, who may exercise a certain measure of discretion in the performance of his duties, including the discretion to make decisions in the ordinary course of the company's business, but who does not participate in the determination of major policies ofthe company and whose decisions are limited by policy standards fixed by senior management of the company; or
Title 12 published on 2015-01-01.
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For a complete list of all Rules, Proposed Rules, and Notices view the Rulemaking tab.
Title 12 published on 2015-01-01
The following are ALL rules, proposed rules, and notices (chronologically) published in the Federal Register relating to 12 CFR Part 225 after this date.


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