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If you have periodontal (gum) disease, it's important for you to know its effects aren't limited to your mouth. A number of studies demonstrate gum disease can affect the rest of your body — and what may be going on elsewhere could likewise stimulate gum disease.
Here are 3 diseases or conditions that seem to share a link with gum disease.
Diabetes. This chronic disease results from the body's inability to interact properly with insulin, the hormone necessary for turning glucose (sugar) into energy, or producing enough of it. There's clear evidence that having diabetes increases your risk of gum disease and vice-versa. If you have diabetes, it's important that you keep it under control for your gum's sake as much as for your overall health.
Cardiovascular disease. Like diabetes, this group of heart and blood vessel diseases has a related characteristic with gum disease: inflammation. This natural function of the immune system limits tissue damage caused by disease or injury. But in both CVD and gum disease, inflammation can become chronic and itself cause damage. Further, some types of bacteria associated with gum disease can contribute to a higher risk of CVD. Minimizing gum disease occurrence with good oral hygiene could positively impact your risk of CVD.
Pregnancy. While certainly not a disease, pregnancy does trigger hormonal changes in the mother that in turn could elevate her risk of gum disease, particularly pregnancy gingivitis. Not only does this pose problems for the mother's teeth and gums, some studies connect gum disease to the increased possibility of early, pre-term birth. A sharper focus on dental care during pregnancy not only benefits the mother but may also be important for the health of the baby.
These aren't the only conditions that can be affected by gum disease: others like osteoporosis, respiratory disease or rheumatoid arthritis also share links with the disease. If you have any systemic condition like these, it pays to be extra vigilant in preventing and treating gum disease.
If you would like more information on periodontal (gum) disease, please contact us or schedule an appointment for a consultation. You can also learn more about this topic by reading the Dear Doctor magazine article "Good Oral Health Leads to Better Health Overall."
Did you make any New Year's resolutions this year? For many of us, these pledges reflect a sincere desire for self-improvement—whether it's in terms of our career, our personal lives, or our health. Yet it isn't always possible to keep every promise we make…and while exercising every day and cutting out desserts are worthwhile goals, they may also be very difficult to maintain. Fortunately, when you resolve to improve your oral health, there are some simple things you can do to help keep your smile looking healthy and bright.
Get Into the Oral Hygiene Habit
Got a minute? How about two minutes, twice a day? If so, you have time to brush your teeth properly. According to the American Dental Association (ADA), brushing twice daily with a soft-bristled brush that fits comfortably in your mouth—and replacing that brush every three to four months—is essential to good oral hygiene. The ADA also recommends flossing once a day to clean all the places where your brush can't reach—like in between teeth and under the gum line. Brushing and flossing are the best ways to maintain good oral hygiene at home.
Think Before You Drink
Here's another way to make a big difference in your oral health: Pass up those sugary and acidic drinks, and choose plain, refreshing water instead. We're talking about regular and diet soda, as well as fruit juice and those so-called "sports" or "energy" drinks. The sugar and acid in these drinks can spell disaster for your teeth: Sugar promotes the growth of bacteria that can cause tooth decay, while acid softens the hard enamel covering of your teeth, allowing cavities to get started. Water, on the other hand, satisfies your body's need for hydration without adding calories or harmful ingredients. That what makes it the best drink for your diet—and your oral health.
See Your Dentist Regularly
There are some jobs best left to the pros—like removing the hardened deposits called "tartar" from your teeth, and checking for tooth decay, gum disease and other oral health problems. We'll take care of all that at your routine dental checkup. Plus, you'll get a thorough cleaning and a chance to "brush up" on oral hygiene techniques that can help you keep your mouth healthy throughout the year.
If you have questions about improving your oral hygiene, please contact our office or schedule a consultation. You can learn more in the Dear Doctor magazine articles "Daily Oral Hygiene" and "Think Before You Drink."
What's the most fun thing about back-to-school time? For many of us, it's the shopping! Those cool backpacks and colorful sneakers put smiles on a lot of young faces. While you're out buying new school supplies and freshening up your kids' wardrobes, why not freshen up their oral health supplies as well?
For example, it might be time for a new toothbrush. The American Dental Association recommends replacing toothbrushes every three to four months, or more frequently if the bristles are frayed. A worn toothbrush won't do as good a job cleaning teeth. And with all of the cartoon characters and superheroes available on toothbrushes, picking one out can be just as fun as choosing a new lunchbox or notebook!
While you're at it, check your kids' supply of dental floss as well. If they've run out, they might not have told you. As important as flossing is, it's not every kids' idea of fun. If you're having trouble getting your kids to use a spool of floss, why not try a disposable little tool made just for flossing? Flossers are super-easy to use, and these, too, come in all kinds of fun shapes and colors.
Here's an important item for the school athletes in your house: a mouthguard. Sports-related dental injuries account for more than six hundred thousand emergency room visits each year. If your child wears braces, a mouthguard may be particularly important. So please contact us about a custom-made mouthguard for your child — or if you have any other questions about oral health and hygiene. And have a safe and healthy return to school!
When it's time for your child to visit the dentist (we recommend around their first birthday), you may want them to see your family dentist. But you might also want to consider another option: a pediatric dentist.
The difference between the two is much the same as between a pediatrician and a family practitioner. Both can treat juvenile patients — but a family provider sees patients of all ages while a pediatrician or pediatric dentist specializes in patients who haven't reached adulthood.
Recognized as a specialty by the American Dental Association, pediatric dentists undergo about three more years of additional post-dental school training and must be licensed in the state where they practice. They're uniquely focused on dental care during the childhood stages of jaw and facial structure development.
Pediatric dentists also gear their practices toward children in an effort to reduce anxiety. The reception area and treatment rooms are usually decorated in bright, primary colors, with toys and child-sized furniture to make their young patients feel more at ease. Dentists and staff also have training and experience interacting with children and their parents to help them relax during exams and procedures.
While a pediatric practice is a good choice for any child, it can be especially beneficial for children with special needs. The "child-friendly" environment is especially soothing for children with autism, ADHD or other behavioral/developmental disorders. And pediatric dentists are especially adept in treating children at higher risk for tooth decay, especially an aggressive form called early childhood caries (ECC).
Your family dentist, of course, can presumably provide the same quality care and have an equally welcome environment for children. And unlike a pediatric dentist who will typically stop seeing patients when they reach adulthood, care from your family dentist can continue as your child gets older.
In the end it's a personal choice, depending on the needs of your family. Just be sure your child does see a dental provider regularly during their developing years: doing so will help ensure a lifetime of healthy teeth and gums.
If you would like more information on visiting a pediatric dentist for your child's dental needs, please contact us or schedule an appointment for a consultation. You can also learn more about this topic by reading the Dear Doctor magazine article "Why See a Pediatric Dentist?"
Is having good oral hygiene important to kissing? Who's better to answer that question than Vivica A. Fox? Among her other achievements, the versatile actress won the "Best Kiss" honor at the MTV Movie Awards, for a memorable scene with Will Smith in the 1996 blockbuster Independence Day. When Dear Doctor magazine asked her, Ms. Fox said that proper oral hygiene was indeed essential. Actually, she said:
"Ooooh, yes, yes, yes, Honey, 'cause Baby, if you kiss somebody with a dragon mouth, my God, it's the worst experience ever as an actor to try to act like you enjoy it!"
And even if you're not on stage, it's no fun to kiss someone whose oral hygiene isn't what it should be. So what's the best way to step up your game? Here's how Vivica does it:
"I visit my dentist every three months and get my teeth cleaned, I floss, I brush, I just spent two hundred bucks on an electronic toothbrush — I'm into dental hygiene for sure."
Well, we might add that you don't need to spend tons of money on a toothbrush — after all, it's not the brush that keeps your mouth healthy, but the hand that holds it. And not everyone needs to come in as often every three months. But her tips are generally right on.
For proper at-home oral care, nothing beats brushing twice a day for two minutes each time, and flossing once a day. Brushing removes the sticky, bacteria-laden plaque that clings to your teeth and causes tooth decay and gum disease — not to mention malodorous breath. Don't forget to brush your tongue as well — it can also harbor those bad-breath bacteria.
While brushing is effective, it can't reach the tiny spaces in between teeth and under gums where plaque bacteria can hide. But floss can: That's what makes it so important to getting your mouth really clean.
Finally, regular professional checkups and cleanings are an essential part of good oral hygiene. Why? Because even the most dutiful brushing and flossing can't remove the hardened coating called tartar that eventually forms on tooth surfaces. Only a trained health care provider with the right dental tools can! And when you come in for a routine office visit, you'll also get a thorough checkup that can detect tooth decay, gum disease, and other threats to your oral health.
Bad breath isn't just a turn-off for kissing — It can indicate a possible problem in your mouth. So listen to what award-winning kisser Vivica Fox says: Paying attention to your oral hygiene can really pay off! For more information, contact us or schedule an appointment for a consultation. You can read the entire interview with Vivica A. Fox in Dear Doctor's latest issue.


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|New Value: $119,950
This property search is part of the Prime Alliance Real Estate Services website. Listing information is courtesy of the WA-NWMLS and Prime Alliance Real Estate Services. Property information is based on available data that may include MLS information, county records, and other sources. Listings marked with this symbol: provided by Northwest Multiple Listing Service, 2020. All information provided is deemed reliable but is not guaranteed and should be independently verified. Properties subject to prior sale or rental Information has not been verified by agent and should be verified by buyer.

subset: commoncrawl


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The video game is not hard to play, fast paced, and a great deal of enjoyable. You might also download some inexpensive Xbox 360 games on the Web! There are a couple of wonderful family friendly Xbox 360 games you are able to play together with your household members and make some outstanding memories.
LEGO Marvel Superheroes
Source : i.ytimg.com
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LEGO Marvel Super Heroes The Video Game
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Chapter 31
"Sam met Samuel Colt when we were trying to find a way to kill Eve." Castiel said holding the letter. "It is addressed to Sam, he should open it."
"How did Sam ever manage to meet Samuel Colt?" Kevin asked. "Colt died in the 1800's right?"
"He drew his last breath on January 10th 1862 ." Balthazar said. "But luckily for Sam and Dean, us angels make Deloreans obsolete when it comes to time travel although it can drain a Seraph pretty quick."
"So you sent Sam back to the 1800's?" Kevin asked.
"Not me. Cassie here did it." Balthazar said. "But why would The Men of Letters pass on a note to Sam?"
"They must have thought that Sam could offer them assistance at some point." Castiel said. "Balthazar, I believe that you should retrieve Sam, since Metatron is aware of the deception Sam's presence should no longer be vital."
Balthazar was suddenly standing there with a stunned Sam Winchester.
"What the?" Sam blinked "Hi Cas, Kevin." He turned to Balthazar "What was that all about?"
"In the immortal words of AOL," Balthazar said taking the note from Castiel and handing it to Sam; "You've got mail."
"Will you settle down Deano? Sam is fine." Gabriel let go of Dean's collar. "Kevin discovered something that needed Sam's attention and Balthazar took him back to the bunker."
Gabriel waited until Dean was looking less like he wanted to kill everything in a 50 foot radius. "Just because Sam is back in the Bat Cave doesn't change what we need to do. Focus Winchester."
"What was so important he couldn't at least say something instead just showing up and grabbing him?" Dean demanded.
"I don't know; but I know we'll find out in due time. For now we just keep an eye on Metatron and find out what he picks up next." Gabriel said as he leaned back in a booth at the roadhouse.
"Gabriel is right Dean." John said from his seat across from Dean next to Gabriel. "I know I told you to look out for your brother but he was still a kid then. He's an adult now, you don't have to hold his hand 24/7."
"Don't have to hold who's hand?" Sam said as he appeared next to the table.
"That was quick." Gabriel noted. "What'd Balthazar moosenap you for anyhow?"
Sam dragged a chair over sitting at the end of the table. "Seems like someone left me a note in the archives."
"What?" Dean didn't even try to hid his confusion. "There was a note for you in that mess of boxes?"
"And I recognized the handwriting." Sam held the note up so Dean could see it. "Samuel Colt."
"So what does it say?" Gabriel demanded.
"I haven't exactly read it yet. But someone in the Men of Letters put this aside along with a letter of their own that whoever found it was supposed to give Colt's note to me or to my descendents."
"So what are you waiting for Sammy, open it."
Greetings Sam,
I pray that box arrived in time to help you. It took me some time to figure out that thingamajig of yours. Is that what you call progress in future times? I'll stick with just writing down what I need to know on paper, it's a lot less convoluted. It's a lot easier for a man to read on paper the words written by another man.
Your visit made me think, Sam. That railroad system, I wasn't going to finish it but I'm thinking that I must have. How else would you have known who and what I am. Only a hunter can know another hunter. So Sam, I'm writing these words for your benefit, trusting that you will understand what to do with them.
"Thingamajig?" Gabriel questioned. "What exactly did you do Sam?"
"I showed him my Blackberry to get him to give me the Colt. We needed it to kill a phoenix and we had a pretty tight deadline." Sam said. "I was in such a hurry to get back to Dean, I forgot it."
"Did you get back in time?" John asked. "To kill the phoenix?"
"Yeah Sam got the colt, I shot the phoenix and had just knelt down to scoop up the ashes when Cas yanked us back." Dean said. "Man that hurt. We were so close."
"And while we were complaining and begging Cas to send us back, there's this knock at Bobby's front door. The FedEx guy is standing there with this shocked look on his face," Sam said with a slight laugh. "He asks for Sam Winchester and handed me this package saying it had been laying around the office forever with a note to deliver it at that date and time. It was from Samuel Colt."
"And inside the box is Sam's brand new Blackberry all beat to hell," Dean continued. "And a jar of phoenix ashes."
"Wow. All that time I spent hunting for that damned Colt, and all I needed to do was get my son to ask Colt to will it to me." John said with a laugh.
"So what does Colt have to say for himself, Bullwinkle?" Gabriel asked.
Sam picked up reading where he had left off.
I finished that railroad, and I was working on the crypt when a couple of fancy Easterners showed up one day real interested in what I was doing. I took them for hunters at first but they didn't know a thing about hunting. What they did know was a lot about the things we hunt. Eventually they told me that they were known as 'Men of Letters'. And that their whole function was collecting lore on creatures, spells, and magic. You name it they knew about it. And they knew about a yellow eyed demon.
While they were concerned about the yellow eyed demon, they were more concerned about a demon they referred to as Abaddon. This demon is bad news, said the only thing worse was Old Splitfoot himself. They have no idea where Abaddon is. I tell you this because you know about the Colt, you know what it can do. That's why you sought me out that day.
Sam I only made 13 bullets for that gun, because you can only make 13 at a time. I learned the secret of that gun in an opium house in Frisco from an old Chinaman. I figured that knowledge would be needed one day, so I wrote it down and gave to the Men of Letters along with all the plans for that railroad devil's trap. I gave it to them to hold onto until Abaddon surfaces again. On that day I pray that you still live or your children.
People spend a lot of time telling me about my guns, they tell me that I will be famous for years to come because of them. They tell me that it's my legacy. I don't much care about that. I don't consider that my legacy because in the end those guns really don't make much of a difference to the world. That crypt in the middle of a lonely cemetery protected by a devil's trap 100 miles across, that Colt I placed in your hands, the knowledge of how to build these things; that's my true legacy. Those are the things that truly make a difference in the world and I give them to you Sam Winchester. You are my heir, I leave this legacy to you.
Samuel Colt.
Sam looked at the second page of the letter which consisted of coordinates, and a name Snake Eyes O'Riley.
Gabriel who had been trying to look at the pages that Sam had been reading suddenly spoke up.
"That's the cemetery the Devil's gate is located in. I'm betting that there's a grave there with the name Snake Eyes O'Riley; but you won't find Snake Eyes in it."
"Dude, you meet Samuel Colt one time and the guy goes out of his way to send you a package a hundred and some years in the future and decides to make you his heir?" Dean noted. "What the hell did you talk about when you went to get that gun?"


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text: Land And PumpsThe bank finally answered the call and the papers have been approved by head office, just awaiting for them to arrive at the branch then we can agree a date to go to the Land Office. Went to the Land Office Thursday afternoon to double checks on the plans that the land was located where we thought. They said they were too busy could we come back in the morning. We went back Friday morning and were in an out in 15 minutes. It is where we thought so hopefully the bank will call today or Monday. The well pump broke again yesterday. having fixed it three times already I decided to replace it, it is obviously not up to the job. Also decided it was time for an automatic pump, and when we are building I'll spare a couple of workers to put a small water tower in for Mama


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MLS #2026846 Official listing information! neighborhood - 111 Free Hill Rd located in Hendersonville TN 37075, priced at $450000, with bedrooms.111 Free Hill Rd Hendersonville TN - MLS #2026846 Close
Commercial for sale in Hendersonville, TN at 111 Free Hill Rd. This commercial is priced at $957,447/acre.
The data relating to real estate for sale on this web site comes in part from the Internet Data Exchange Program of RealTracs Solutions. Real estate listings held by brokerage firms other than The Ashton Real Estate Group of RE/MAX Advantage are marked with the Internet Data Exchange Program logo or thumbnail logo and detailed information about them includes the name of the listing brokers.
Disclaimer: All information is believed to be accurate but not guaranteed and should be independently verified. All properties are subject to prior sale, change or withdrawal.
Copyright 2020 RealTracs Solutions.
Listing information last updated on January 23rd, 2020 at 11:25am CST.


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Browsing MBA-HCM Theses and Dissertations (2018) by Author "Gone, Michael"
Now showing items 1-1 of 1
Effects of co-payments on utilization of healthcare services at a private out-patient facility: a case study of AAR healthcare Kisumu Out-patient Centre Gone, Michael (Strathmore University, 2018)Rising cost of healthcare is a concern worldwide. In Kenya, this has seen insurance companies report losses despite increase in premiums. In order to control the rising costs, insurance companies have introduced co-payments ...


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How were you unlucky enough to be born in China?
From China Digital Space
Shéi jiào nǐ búxìng shēng zài Zhōngguó le? 谁叫你不幸生在中国了?
Question posed to a reporter by physicist and "crusader against unscientific thinking" He Zuoxiu, He Zuoxiu; rhetorical question for Chinese citizens hurt by corruption in the name of progress.
He Zuoxiu, a member of the Chinese Academy of Science, advocated for former president Hu Jintao's concept of a "[[Use the Internet scientifically|scientific outlook on development]" (kēxué fāzhǎn guān 科学发展观). In a 2005 interview with a reporter for Southern People's Weekly, He was asked about deaths in coal mines. The reporter offered that more than o100 people die every day in China's coal mines. He countered that a much greater number died in car accidents. When pressed by as to whether the coal mine deaths were the avoidable results of government corruption, He denied this and claimed instead that the deaths were necessary byproducts of poverty. He shot back at the reporter, "How were you so unfortunate as to be born in China?" (literally "Who told you to be born unluckily in China?"), adding, "In the process of development some sacrifices are unavoidable."
For many netizens, He's excoriation of the reporter symbolizes the emotional detachment with which the government pursues economic growth, the logical but soulless endpoint of "scientific development."
This retort to scientific development has remained in currency after Hu Jintao left office. For instance "How were you unlucky enough to be born in China?" has appeared as a sad comment on Liu Lingli, a young teacher who was fired for "absenteeism" while she was being treated for cancer. She passed away in August 2016.


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Rain Drops
Literary Supplement
Reglar Wiglar #3, 1994
Welcome to the first addition of what I can only hope will be a long and successful career for the Rain Drops Literary Supplement. Regrettably, I had not a modicum of time to contribute any of my own work as I was so dilligently working on the supplement as a whole--contacting various writers, poets and procuring the works of the legendary Charles Franklin Buchanan. The latter was a task accomplished with great facility, as Mr. Buchanan is deceased. Getting young writers and poets to contribute was a little more difficult, however. I would think that young artists would spring at the chance to be involved with a publication such as this. Rain Drops is, after all, affiliated with the Reglar Wiglar. But alas, none could meet my very lax deadline. Apparently, in today's hectic and poetic world, six months to complete a three stanza poem is too taxing on the creative mind and too stifling on the creative spirit. Whatever the case, or excuse, here is Rain Drops, what little of it there is. I hope you enjoy it or at least appreciate it and the work I've done, or at the very least, read some of it or maybe just read the titles and give a little contemplative "hmmmmmmm" for my sake, please—Jayne Wayne
C.F. Buchanan: Hard to the Core
The man that was, the legend that is, the late Charles Franklin Buchanan will never die. Too much legacy has been left to us for his memory to ever fade. Often thought to be the father of Hard Core Fiction, C.F. Buchanan wrote for over fifty years, leaving behind a body of work that fills three volumes of poetry, essays, and fiction. A political satirist and a sharp, unrelenting social critic, Buchanan remains unchallenged as one of America's greatest writers and crankiest old drunks.
The Forgetter of Things
The rain urinated from the sky like a steady stream from a back alley drunk on garbage cans, concrete and broken city dreams. Cracker box laughter rifled through the avenue, piercing ears in its wake, sounding off like a bumper car chainsaw, waterfall screams from some hormone screw cup gushing like a bucket. Weeds, forgotten like beanbag chairs, collecting the ages in dusty neglect and discarded shoe trees whined for a little more than what they were getting. Weeds. Fucking weeds.
Booker staggered through the front door of the Back Door Tavern, wood creaking like a ruptured spleen in the night. "Goddamn the machine" were the only trio of words he could udder out of his crooked vain lips, flesh sagging like a sack of dead rats in the river. Syllables splashed on the cobblestone like dishwater--dirty and mean, filth from a thousand years of toilage and internal judgement. Bitter, then silent. Who could understand his pain? Not she. She who twisted, mangled his pumping organ rendering it incapable of any emotion with the ragged exception of rage, putrid and unholy, unclean like a bus station floor, grime, sin and filmy dirt of an unknown, undesired, unforgiving origin. You clean up this mess, man. This is your mess, man.
Booker swayed, unsteady like a ball bearing. External orbiting around playground perverts, sick and twisted. Mind? No, I insist, happy as only a molester of children can be, a forgetter of birthdays and annivesaries, unfit for the society that produced him, that preys on the weak and the sick and the tardy. You, man, you are the forgetter of things. You, man. You!
Then hunger. A new emotion sensation pelting, plastering, toppling, grating then chiding his gut wrenching bellow, balk and stammer in the sickly train light of the captured moon. Profess to know nothing. Stir, pillage, plummet, cut your gains and imitate your losses. Punch shock your sweaty deceptive dreams. Reach and shatter drive them. Watch them flutter, bewitched, hung like a horse thief in the night. Climb, grab and cling to the things you hate yourself for hating. Tamper with the pulse of the jiggly night. Be the beggar mouse princess, the pacifier of things that can not be. Son of a tax payer. Son of a brick layer. God damn the son of man.
I gotta get some sleep Booker imagined he thought to himself as crunchy knees consummated some sacred ritual with cold concrete coated earth. And then silence more soundless than the bottom of some gigantic cup of cool. Blackness to rival the dark side of Pluto's moon under an oak tree old as Jesus.
"Forgetter of Things" appeared as "Cup of Cool" in Reaction Magazine ©1990 C.F. Buchanan. This version excerpted from the novel Dirty Deeds Done for Free originally published 1991 by Big Horny Publications.
Under the Pus Filled Moon
Under the pus filled moon
In soaking trousers desire lurks
From prison walls to urine stalls
Go bretheren of man
Under the pus filled moon
"Under a Pus Filled Moon" originally published in Graft Poetry Anthology. ©1981 C.F. Buchanan
Legalize Crack, Give Guns to School Children and Kill the Homeless
The press fiddles have been at it again. Fired up with their pearl lighter football games, strutting around with toothache smiles and sloe gin grins, tooling for some asphalt dumb dickey, too loaded on jag hornet bullshit and pocket swivel to cop a sense of the times at hand. It burns me like a dust bottle scalp spray when clear, clean stone demographics of where the lagheads dwell and are drowned in their own self pity are traded like a Tuscon winter sled ride for some two bit information super highway that never knew what hit it.
Leaving you and me to fight off the overzealous zealots, despots, crackpots, hacks and other politicians bleeding like an ulcer in the stomach of a nation too crushed on candy-coated sound bites and spin doctored keep-you-in-a-can cucharacha baby, "ya no puedo caminar."
If the sinners keep sinnin' then the devil keeps a-winnin' and we know better than to let a bunch of fool proof, off the bottle into the pan hooligans with jack boots and ignorant pants flailing in the heat like so many unborn burdens we least desired most. There's no easy way out of the twisted forrest this time, Cinderella. We all gotta face the music even if it is your own twisted bodily nosing off about the price we pay for the people we lay.
So give crack to school children. It couldn't hurt their tiny souls anymore than the lies we pack into their knowledge vessels. Pack the pipe and clean out the filth that a plumber and a can of 'fuck you up' couldn't cope with in a termite's ass. Unpatriotic? Maybe. UnAmerican? Never.
Yes, the press fiddles have been eagerly at it again, conjuring up these mad, yet benign messages forcasting doom for the damned. Stone Demographics don't lie. You can crack a hundred eggs in a bucket and not break one yoke but that doesn't change a thing. Put that in your screw cup and drink it.
Moronic men have made meaning out of the meatless items on the menu, not knowing, not caring about the jellyfish stigma of unwashed heathen at the gate, prepared to do battle with the neverending, the unphotogenic, the cursed multitude who show no gratitude.
*exceperted from a lecture at University of Wisconsin, Madison in March 1990. ©1990 C.F. Buchanan
© 1993-2020 Reglar Wiglar Magazine


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5S rRNA is a highly conserved ribosomal component. Eukaryotic 5S rRNA and its associated proteins (5S rRNA system) have become very well understood. Giardia lamblia was thought by some researchers to be the most primitive extant eukaryote while others considered it a highly evolved parasite. Previous reports have indicated that some aspects of its 5S rRNA system are simpler than that of common eukaryotes. We here explore whether this is true to its entire system, and whether this simplicity is a primitive or parasitic feature.
Methodology/Principal Findings
By collecting and confirming pre-existing data and identifying new data, we obtained almost complete datasets of the system of three isolates of G. lamblia, two other parasitic excavates (Trichomonas vaginalis, Trypanosoma cruzi), and one free-living one (Naegleria gruberi). After comprehensively comparing each aspect of the system among these excavates and also with those of archaea and common eukaryotes, we found all the three Giardia isolates to harbor a same simplified 5S rRNA system, which is not only much simpler than that of common eukaryotes but also the simplest one among those of these excavates, and is surprisingly very similar to that of archaea; we also found among these excavates the system in parasitic species is not necessarily simpler than that in free-living species, conversely, the system of free-living species is even simpler in some respects than those of parasitic ones.
The simplicity of Giardia 5S rRNA system should be considered a primitive rather than parasitically-degenerated feature. Therefore, Giardia 5S rRNA system might be a primitive system that is intermediate between that of archaea and the common eukaryotic model system, and it may reflect the evolutionary history of the eukaryotic 5S rRNA system from the archaeal form. Our results also imply G. lamblia might be a primitive eukaryote with secondary parasitically-degenerated features.
Citation: Feng J-M, Sun J, Xin D-D, Wen J-F (2012) Comparative Analysis of the 5S rRNA and Its Associated Proteins Reveals Unique Primitive Rather Than Parasitic Features in Giardia lamblia. PLoS ONE 7(6): e36878. https://doi.org/10.1371/journal.pone.0036878
Editor: Paul Wrede, Charité-Universitätsmedizin Berlin, Germany
Received: February 1, 2012; Accepted: April 13, 2012; Published: June 7, 2012
Copyright: © 2012 Feng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Natural Science Foundation of China (30830018; 31172081)and the Knowledge Innovation Program (KSCX2-EW-J-23). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
5S rRNA is a highly conserved component of large ribosomal subunit in all organisms, excepting only certain mitochondrial systems [1]. In prokaryotes, 5S rRNA is generally transcribed in a common transcription unit with 16S and 23S rRNAs, while in most eukaryotes, 5S rRNA gene is located separately from 18S, 5.8S and 28S rRNA genes and is transcribed alone by RNA polymerase III (RNA pol III) with the participation of a specific transcription factor IIIA (TFIIIA). Here TFIIIA not only acts as an essential transcription factor by binding to the 5S rRNA gene but also combines with 5S rRNA itself to form a 7S ribonucleoprotein (RNP) particle to stabilize the rRNA and facilitate its nuclear export [2], [3]. Ribosomal protein L5 is also known to bind specifically to 5S rRNA and to be involved in its cytoplasmic-nucleolar transport. In eukaryotes, prior to ribosome assembly, the 5S rRNA transcript changes binding partners, trading TFIIIA for L5, and is then escorted to the nucleolus by the latter [4]. In the present work, we refer to 5S rRNA and these associated proteins as the 5S rRNA system. From the above, we find that the components and function of this system are very clear, making it a suitable candidate for investigation across diverse organisms for the purposes of a comparative evolutionary study.
Giardia lamblia is one of the most widespread intestinal protozoan parasites in the world. It was once thought to be the most primitive extant eukaryote because it has many so-called primitive molecular and cellular traits [5] and branches out very early from the eukaryote trunk on many phylogenetic trees [6]–[10]. But, actually Giardia's evolutionary position has been under controversy since the proposition of its being extreme primitive eukaryote [11], especially after the discovery of its mitochondrial remnant organelle mitosome [12]. Recently, phylogenomic analyses based on large phylogenomic datasets have placed G. lamblia within excavate, one of the three primary divisions of eukaryotes [13]. However, no matter it is primitive or not, it is amazing that it was reported that in this organism no 5S rRNA was detected in either experiments or BLAST searches against its genome database [14], [15], since 5S rRNA is so ubiquitous and highly conserved in both prokaryotic and eukaryotic cells. For a long time, there were no 5S rRNA annotations in the G. lamblia genome database before we began the present study. Later, 5S rRNA genes, though there were only eight copies, far fewer than the number seen in most common eukaryotes, were annotated in G. lamblia genome database [16]. Studies on some G. lamblia 5S rRNA-associated proteins revealed that they are much simpler than those of typical eukaryotes. For example, only one subunit specific to RNA pol III, called C34, was found, and none of the transcriptional factors of RNA polymerase III (TFIIIA, TFIIIB, or TFIIIC) were found either. There were only two components of TFIIIB (TBP and BRF) identified in the G. lamblia genome database [17]. But it is unknown whether the entire G. lamblia 5S rRNA system is simpler than that of common eukaryotes yet.
Some authors consider the simplicity of many aspects of G. lamblia, including genomic reduction, to be the results of parasitic degeneration rather than primitive traits [16], [18]–[20]. We doubt that this is the case for certain aspects of this organism, such as 5S rRNA system, which have no direct relationship with parasitic lifestyle but are rather involved in the fundamental functions of a eukaryotic cell. To confirm this, by collecting published and identifying new data of 5S rRNA system, we compared the G. lamblia 5S rRNA system to those of two parasitic species (Trichomonas vaginalis and Trypanosoma cruzi) and one free-living species (Naegleria gruberi), which here served as the representatives with genome database in other three subgroups of excavate, respectively, and also to those of archaea and common eukaryotes. In addition, the genome databases of two other G. lamblia isolates, assemblage B isolate GS and assemblage E isolate P15, are now available [21], [22]. In this way, we identified their 5S rRNA system to confirm whether the simplicity of the 5S rRNA system is a common trait among different G. lamblia isolates.
Materials and Methods
Data regarding genomes and the annotated transcripts and proteins of G. lamblia assemblage A isolate WB, B isolate GS, and E isolate P15 were downloaded from the GiardiaDB (http://giardiadb.org/giardiadb/). Genomic data regarding T. vaginalis (http://trichdb.org/trichdb/), T. cruzi (http://tritrypdb.org/tritrypdb/), and N. gruberi (http://genome.jgi-psf.org/Naegr1/Naegr1.home.html) were downloaded from their respective databases.
Data Collection and Identification
We collected previously reported data on the 5S rRNA system, including the 5S rRNA gene, RNA pol III, associated transcriptional factors, and L5, in G. lamblia (including isolates WB, GS, and P15), T. vaginalis, T. cruzi, and N. gruberi. They are summarized as follows: 1) The 5S rRNA gene and its promoters: Eight 5S rRNA genes were reported in the genome database of G. lamblia WB and six in the P15 database [16], [22]. The 5S rRNA gene and its TATA box and internal control region (ICR) were reported in T. vaginalis [23]. 5S rRNA genes and their ICRs were identified in T. cruzi [24]; 2) transcriptional factors and RNA pol III-specific subunits: a TFIIIB with only two components and a RNA pol III with only the specific subunit, C34, were previously reported in G. lamblia WB [17]; 3) L5 sequences of G. lamblia (including WB, GS, and P15), T. vaginalis, T. cruzi, and N. gruberi were all obtained through the Entrez system at NCBI (http://www.ncbi.nlm.nih.gov/sites/gquery).
The data not already published were identified as follows:
5S rRNA Gene
An in silico search for the 5S rRNA genes of G. lamblia GS and N. gruberi was performed using the BLASTn program against the G. lamblia GS genome database and N. gruberi genome database [21], [25]. The 5S rRNA gene sequences from prokaryotes and eukaryotes that were used as query sequences were retrieved from the 5S rRNA database (http://www.man.poznan.pl/5SData) [26]. BLAST search default parameters were modified to reduce stringency: the penalty for a gap or a mismatch was changed from 3 to 1 to enable us to identify the 5S rRNA gene. The 5S rRNA genes of G. lamblia WB and P15 were used as queries to search for 5S rRNA gene against the G. lamblia GS genome database by using the BLAST program (http://giardiadb.org/giardiadb/).
Data regarding the internal control region (ICR) and TATA box were found in the G. lamblia 5S rRNA genes in the following manner: For ICR, the consensus sequences of its three elements: Box A [5′-N(G/C)(C/T)(C/T)AANCNNNNNNN-3′], intermediate element (IE) [5′-(C/G)NN(G/A)(G/A)N-3′], and Box C [5′-NNG(G/A)TGGGNG(T/A)CCN(C/T)NNG-3′] were searched manually within the G. lambia 5S rRNA genes. For the TATA box, its highly conserved core sequence (5′-TATAAA-3′) was used as a consensus sequences to analyze the upstream sequences of G. lamblia 5S rRNA genes. Because of the flexible position of the TATA box, an AT-rich region, being relative to the transcriptional start site in G. lamblia, the entire 60 bp upstream region of the transcriptional start site of G. lamblia 5S rRNA genes were analyzed. Using these same methods, the ICR of 5S rRNA gene in N. gruberi and TATA boxes in the 5S rRNA genes of T. cruzi and N. gruberi were identified.
No TFIIIA had previously been identified in G. lamblia WB by homolog search against its partial genome assemblies [17]. Once the fully sequenced G. lamblia WB genome database was available, we tried to identify TFIIIA via BLASTp and PSI-BLAST searches against the database again. Considering the high primary sequence divergences of TFIIIA among different species, we used the C2H2 motif of TFIIIA as query. The C2H2 motif-containing sequences were obtained from the PROSITE database (release 32 of the PROSITE pattern ZINC-FINGER_C2H2, Accession No. PS00028). Then the best hits were filtered by the presence of C2H2 zinc finger domains using the Interproscan program [27]. TFIIIA was identified in G. lamblia GS and P15, T. vaginalis, T. cruzi, and N. gruberi by BLASTp and PSI-BLAST searching against their corresponding genome databases.
TFIIIB, TFIIIC, and Specific Subunits of RNA Pol III
The data regarding TFIIIB, TFIIIC and specific subunits of RNA pol III in G. lamblia were reported previously by BLAST searching against the G. lamblia WB partial genome assembly [17]. In this work, we again found them in the fully sequenced genome database. Saccharomyces cerevisiae sequences of subunits of TFIIIB and TFIIIC and of the specific subunits of RNA pol III were used as queries to BLASTp search against genome databases of all the three G. lamblia isolates, T. vaginalis, T. cruzi, and N. gruberi, to get putative homologs. Then the putative homologs were assessed using domain information in the Pfam database (http://pfam.sanger.ac.uk) [28]. Another BLASTp search was performed, this time against the non-redundant (nr) NCBI protein database. Sequences lacking characteristic domains of the given proteins or indicating other proteins were discarded. For proteins for which no homologs could be found by BLASTp search, at least five iterations of PSI-BLAST search was carried out online to identify putative homologs. Then these putative homologs were confirmed by Pfam domain analysis and BLASTp against the NCBI nr database, as described above.
RT-PCR and Sequencing
To examine the transcription activities of the G. lamblia 5S rRNA genes, reverse transcription polymerase chain reaction (RT-PCR) experiments were carried out as follows: Small RNAs (≤200 nucleotides) were extracted from G. lamblia WB cells using a mirVana™ miRNA Isolation Kit (Ambion, Austin, TX, U.S.) according to the manufacturer's instructions. Then the small RNAs were polyadenylated at 37°C for 30 min in 60 µl of reaction mixture with 500 ng small RNAs, 3 mM rATP, 6 µl 10×Buffer and 15 U E. coli poly(A) polymerase (New England BioLab), and the poly(A)-tailed RNAs were recovered by phenol/chloroform extraction and ethanol precipitation. Finally the purified RNAs were used as templates for RT-PCR, using a RNA PCR Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China.) according to the manufacturer's instructions. The gene-specific primers were designed according to the sequence of our identified G. lamblia 5S rRNA genes. Primers ((Pfr 1∶5′-TCggCCATCCTACggCggAAACT-3′; Pfr 2∶5′-CCCACgACgTCTCCgATCgCAgT-3′) were synthesized by Sangon (Sangon Ltd., Shanghai, China).
The purified products of these RT-PCR experiments were cloned into PMD 19-T vectors (TaKaRa). Twenty clones were selected at random and sequenced at Sangon Ltd.
Identification of 5S rRNA Genes in G. lamblia (WB, GS, and P15) and N. gruberi
Our BLASTn search included modified parameters to reduce stringency. It confirmed that there are only eight 5S rRNA genes located separately in the G. lamblia WB database and six in the P15 database. This is consistent with the annotations in these two databases [16], [22]. Based on the genome sequence, seven of the eight G. lamblia WB 5S rRNA genes each were found to have a putative 5S rRNA coding region 117 bp in length while the other had an extra 15 bp insert within the coding region. However, in our RT-PCR and sequencing experiments, the products detected were only from the 117 bp genes. Thus, the gene with the extra insert might not be transcribed.
No putative 5S rRNA gene was identified in the G. lamblia GS genome database upon the retrieval of various 5S rRNA sequences from the 5S rRNA database as queries to BLASTn search against the genome database. However, one hit (Contig ID: ACGJ01000259), which was found to possess a 36 bp sequence sharing 100% identity with the 5′ terminal region (from+1 bp to 36 bp) of 5S rRNA genes of G. lamblia WB and P15 was found when the 5S rRNA genes of the two isolates were used as queries (Figure 1). The region in contig ACGJ01000259 (309 bp long) sharing 100% identity with 5S rRNA gene (from+1 bp to 36 bp) of G. lamblia WB and P15 was found at the end of this contig (from 274 bp to 309 bp). Moreover, a 79 bp RNA fragment sharing 99% identity with the 3′ terminal region (from +39 bp to +117 bp) of G. lamblia WB 5S rRNA was once reported in G. lamblia GS [21]. In this way, the lack of a G. lamblia 5S rRNA gene in the G. lamblia GS genome database is most probably due to the numerous gaps in the database and the small number genes in this organism.
r0002, r0005, r0006, r0007, r0011, r0015, r0026, and r0008 represent the eight G. lamblia WB 5S rRNA genes. ACGJ01000259 (274–309) represents sequences from residue 274 to 309 of contig ACGJ01000259 in the G. lamblia GS genome. Box denotes the putative partial 5S rRNA gene identified in G. lamblia GS sharing 100% identity with 5′ fragment of 5S rRNA gene in G. lamblia WB and P15.
Using the BLASTn program to search against the N. gruberi genome database, we found 279 potential 5S rRNA genes (Table S1).
Comparisons of 5S rRNA Genes among G. lamblia, and Other Three Excavates, Common Eukaryotes, and Archaea
According to the above identified 5S rRNA genes in G. lamblia and N. gruberi, and the reported many copies of 5S rRNA gene in T. vaginalis and T. cruzi [23], [24], it can be said that in contrast to common eukaryotes and the other three investigated excavates, G. lamblia had a very small copy number of 5S rRNA genes and no tandem 5S rRNA gene regions, suggesting that it is very similar to archaea (Table 1).
Analysis on putative promoter elements revealed that there are no special relevance to the intermediate element [IE: 5′-(C/G)NN(G/A)(G/A)N-3′], the Box A [5′-N(G/C)(C/T)(C/T)AANCNNNNNNN-3′], or Box C [5′-NNG(G/A)TGGGNG(T/A)CCN(C/T)NNG-3′] of the ICR in any part of the G. lamblia WB or P15 5S rRNA genes. This indicates that these genes do not possess ICR and would not bind TFIIIA specifically during transcription, as found in common eukaryotes. Our analysis showed that presence of ICR in the 5S rRNA genes of N. gruberi (Figure 2). The presences of ICR have been reported previously in the 5S rRNA genes of T. cruzi and T. vaginalis [23], [24]. This suggests that the three other excavates, both parasitic and free-living, are similar to common eukaryotes in this respect.
) The putative ICR promoter elements identified in N. gruberi 5S rRNA gene. The TATA box is denoted in bold. Underlined sequences refer to the putative ICR. The proposed first transcribed base (T) is positioned with 1 and the putative last nucleotide of the mature molecule is shown circled (T in position +118). Empty boxes indicate T-runs as putative termination sequences. B) No TATA box could be identified in the T. cruzi 5S rRNA gene.
However, although no canonical TATA box was found in G. lamblia 5S rRNA genes, we have identified a 16 bp AT-rich region between −58 and −43 bp upstream of the transcriptional initiation site of these genes, whose consensus sequence is 5′-AA(T/C)A(C/T/A)(A/G) C(A/T)(G/A)(T/A)AT(G/T)(T/A)A(T/C)-3′ (Figure 3). Previous studies have indicated that the TATA boxes of G. lamblia genes are not as canonical as those of common eukaryotes. Rather, they vary with respect to their sequence, length, and exact position relative to the transcription start site [29]–[33]. Our analysis also showed a TATA box in the 5S rRNA genes of N. gruberi but not in T. cruzi (Figure 2). T. vaginalis has been reported to have TATA box in its 5S rRNA genes [23] (Table 1).
The regions between −58 and −43 bp upstream the transcriptional initiation site of G. lamblia WB 5S rRNA genes all contain an AT-rich region (denoted by box), which might be the TATA-like box of G. lamblia WB 5S rRNA gene.
Identification and Comparison of Transcriptional Factors and Specific Subunits of RNA Pol III
TFIIIA was found in T. vaginalis but not in T. cruzi, N. gruberi, or the three G. lamblia isolates upon BLASTp or PSI-BLAST search against their corresponding genome databases. We then used a C2H2 motif as a query for another BLASTp search against the G. lamblia WB genome database to confirm whether TFIIIA is really absent from it. After the BLASTp search, we found 1,959 best hits. Of these, 102 were predicted to be putative zinc finger proteins, and 18 out of these 102 proteins were further predicted to be C2H2-type zinc finger proteins. But 17 out of the 18 proteins each contained only 1–2 zinc finger repeats, and the other one (ORF: 17003) had a total of four repeats. In common eukaryotes, the number of TFIIIA finger repeats is usually nine. None of the 18 C2H2-type zinc finger proteins were annotated as TFIIIA in the NCBI database (Table S2).
M, marker.
Our investigation further confirmed that this organism lacks TFIIIB and TFIIIC, but two components of TFIIIB were present in G. lamblia WB as previously reported. In G. lamblia GS and P15, similarly, no TFIIIB or TFIIIC was found, but two components of TFIIIB were identified. However, both TFIIIB and TFIIIC were found in all the other three investigated excavates, T. vaginalis, T. cruzi, and N. gruberi (Table S3).
Of the five specific subunits of RNA pol III (C17, C31, C34, C53, and C82), two (C34 and C82) were identified in G. lamblia WB, GS, and P15 by our PSI-BLAST. Of these, only one, C34, had been reported in G. lamblia WB before this work [17]. Two, C34 and C53, were found in T. cruzi; three, C34, C53, and C82, were identified in T. vaginalis; and four, C34, C53, C82, and C17, were found in the free-living species N. gruberi (Table S3).
Examination of Transcription of G. lamblia 5S rRNA Gene
Our RT-PCR experiment on the G. lamblia WB 5S rRNA genes showed positive results (Figure 4). The sequencing results indicated the RT-PCR products were the exact transcripts of the seven G. lamblia WB 5S rRNA genes with the normal length (117 bp), while no transcript corresponds to the other one which has a 15 bp insertion. Therefore, the seven normal-length G. lamblia WB 5S rRNA genes were found to be actively transcribed while the other was not.
By collecting and confirming the existing data and identifying new data, we were able to construct almost complete datasets of the 5S rRNA systems of the three sequenced isolates of G. lamblia and three other excavates. Therefore, in this study, we were able to comprehensively compare each aspect of the 5S rRNA system among them and with those of archaea and common eukaryotes.
In the genomes of common eukaryotes, 5S rRNA genes are usually arranged in tandem arrays of several hundreds and thousands of copies in several different rDNA regions. The total gene copy number varies widely by species. Humans have 1700–2000 copies and Xenopus oocytes have ≈20,000 [34], [35]. Excavates, Crithidia fasciculata have ≈250, T. vaginalis and T. cruzi each have hundreds and thousands of copies [23], [24], [36]. There are 279 putative 5S rRNA genes in N. gruberi. However, our study has indicated that in all the three sequenced G. lamblia isolates, only a few copies of 5S rRNA genes exist. The total gene copy number in G. lamblia is unexpectedly small, and the genes are not arranged in tandem. This is not consistent with the pattern observed in common eukaryotes and in other excavates (both free-living and parasitic) but rather with those in archaea. On the other hand, the G. lamblia 5S rRNA genes are located far from the 18S-5.8S-28S rRNA gene unit, as found in common eukaryotes, suggesting they are also transcribed independently by RNA pol III.
In this study we found that there are no ICR but rather a gene-external TATA-like box in each G. lamblia 5S rRNA gene. Most eukaryotes have both [37]. In the other three excavates studied, both well defined gene-external TATA box and gene-internal ICR appear in T. vaginalis 5S rRNA genes [23], and both were also found in 5S rRNA genes of N. gruberi in the present study, however, there is only gene-internal ICR in T. cruzi 5S rRNA genes [24]. The characteristics of 5S rRNA gene promoters in these excavates suggests which kind of promoter appears in a species has nothing to do with its lifestyle. However, the results of our study indicate that the G. lamblia 5S rRNA gene promoter might be a very primitive, and similar to that of archaea. Moreover, this might further supports the idea that during evolution of eukaryotes, the gene-external TATA box promoter of the 5S rRNA genes might have been inherited from archaea and emerge very early in the evolution of eukaryotes, while the gene-internal ICR arose at later point after the divergence of G. lamblia from the eukaryotic trunk.
Usually, three transcription factors are involved in the transcription of 5S rRNA by RNA pol III in common eukaryotes, and TFIIIA is the only one specific to this process. The binding of TFIIIA to the ICR of 5S rRNA gene is the first step of the transcription of this gene. This allows the recruitment of TFIIIC, TFIIIB, and RNA pol III [38]. TFIIIA also combines with 5S rRNA to export it from the nucleus to the cytoplasm. Up to now, knowledge about TFIIIA has mainly come from studies of higher eukaryotes, and no TFIIIA has ever been reported in protists. One previous study of G. lamblia reported finding no TFIIIA [17]. Considering that this previous study was based on partial G. lamblia genome assemblies, the present study used the complete G. lamblia genome databases and relatively conserved C2H2 domain of TFIIIA as query to search TFIIIA, but still identify no homolog to the typical eukaryotic TFIIIA in the three isolates of G. lamblia. Our sequence analysis also revealed that the absence of ICR in the G. lamblia 5S rRNA gene, which probably also implies the absence of TFIIIA. These findings further confirm the absence of TFIIIA in Giardia, and suggest that G. lamblia 5S rRNA transcription is TFIIIA-independent. Previously, TFIIIA-independent 5S rRNA transcription in eukaryotes was only reported in Yarrowia lipolytica, where 5S rRNA gene is co-transcribed with tRNA [39]. But no G. lamblia 5S rRNA genes have a neighboring location with tRNA gene as in Y. lipolytica according to our investigation. Furthermore, there is no homologs of TFIIIB and TFIIIC in G. lamblia [17], which was also further confirmed in the present study. These findings may indicate that the initiation mechanism of G. lamblia 5S rRNA transcription is different from those of all eukaryotes studied. Although it does not have TFIIIB, G. lamblia possesses two important subunits of TFIIIB: TATA box binding protein (TBP) and TFIIB-related factor (BRF) [17]. In archaea, TBP and TFB can mediate the initiation of basal transcription [40]. Archaeal TFB and eukaryotic BRF are evolutionarily related by sharing a common ancestry [17]. For this reason, the initiation mechanism of G. lamblia 5S rRNA transcription should be similar to that of archaea, specifically, with respect to without the participation of TFIIIA, TFIIIB, and TFIIIC but including TBP and TFB/BRF. As for the other three investigated excavates, both TFIIIB and TFIIIC exist in all of them, while TFIIIA only exists in the parasitic excavate T. vaginalis and not in the free-living N. gruberi. This means that parasitism does not necessarily lead to the reduction of the transcription factors of RNA pol III. Therefore, the simplicity of the G. lamblia transcription factor system should not be interpreted as necessarily the results of parasitic degeneration but most probably as a primitive feature of this organism.
The three eukaryotic RNA polymerases (pol I, II, III) all have both common subunits and subunits specific to each of themselves, whereas G. lamblia RNA pol III has been reported to possess all the common subunits but lack all specific subunits except for C34 [17]. However, besides C34, we found the fully sequenced G. lamblia genome to include another specific subunit, C82. In yeast, subunit C34 was shown to interact with subunits C82 and C31 to form a subcomplex functioning in the initiation of RNA pol III transcription [41]. Subunit C34 and C31 were shown to play a key role in determining the initiation specificity of RNA pol III transcription [42], [43]. Archaeal RNA polymerase has recently been reported to have one ortholog to C34, which might be involved in the transcription of non-coding RNAs [44]. Our investigation revealed the presence of C34 and C82 in all the investigated excavates (except T. cruzi, which lacked C82, probably due to secondary loss), while C31 was absent from all of them. These mean that C34 might have been inherited from archaea and emerged very early in the evolution of eukaryotes, while C31 arose at least after the divergence of excavate from the eukaryotic trunk. Lacking C31, the RNA pol III of excavate might carry out the specific initiation of RNA pol III transcription using only C34 and C82. This is similar to the initiation of non-coding RNA transcription only with C34 in archaea. However, the parasitic excavates T. vaginalis and T. cruzi also have another more specific subunit that is absent from G. lamblia, C53. Therefore, the similarity of G. lamblia RNA pol III to those of archaea also seem not to be due to parasitic degeneration but to be the very primitive traits of this organism.
Since TFIIIA also functions as a 5S rRNA transcript chaperone required for the nuclear-cytoplasmic transport in common eukaryotes [3], [45], without TFIIIA, the 5S rRNA nuclear-cytoplasmic transport in G. lamblia must be unique. Whereas, the presence of 5S rRNA-associated protein L5 in G. lamblia, however, suggests that the mechanisms of G. lamblia 5S rRNA cytoplasmic-nucleolar transport and incorporation into large ribosomal subunit are similar to that of common eukaryotes. No TFIIIA has yet been reported in archaea, but L5 protein has been reported [46], [47]. The G. lamblia 5S rRNA transport system can then be said to be similar to that in archaea. The fact that TFIIIA is present in the parasitic excavate T. vaginalis but absent from the free-living N. gruberi further suggests that the absence of TFIIIA in G. lamblia is not necessarily due to parasitic degeneration but most probably to the primitive nature of this organism.
In summary, our results reveal a unique 5S rRNA system in G. lamblia: On one hand, some of its features, such as the separated location of the 5S rRNA genes and the transcription of 5S rRNA genes by RNA pol III, are consistent with that seen in common eukaryotes. On the other hand, many other traits, such as the lack of a tandem 5S rRNA gene region, the low gene copy number, the relatively simple transcription system (lacking the participation of TFIIIA, TFIIIB, and TFIIIC but inclusion of TBP, BRF, the RNA pol III with only two specific subunits, C34 and C82), and the TFIIIA-independent but L5-dependent 5S rRNA transport and incorporation system, are much simpler than those of most eukaryotes and very similar to those of archaea. Comparisons of the entire 5S rRNA system among the three sequenced G. lamblia isolates and with the other three investigated excavates showed 1) All the three G. lamblia isolates harbor the same simplified 5S rRNA system, which is the simplest one among all the investigated excavates in almost every aspect of the system; 2) parasitic species are not definitely simpler than that of free-living ones, conversely, the free-living species can be simpler in some reaspects of the system, such as transcription factors (Table 1). In fact, as an obligate parasite, though not an excavate, Encephalitozoon cuniculi was also compared to G. lamblia by us. We found the 5S rRNA system of G. lamblia is to be simpler than that of E. cuniculi, especially in the aspect of transcription factors (Table S4). In this way, the simplicity of G. lamblia 5S rRNA system should not be considered due to parasitic degeneration. If this is not ture, then it would become difficult to explain why the system was definitely degenerated to be an archaeal form.
In conclusion, G. lamblia represents an ancient primitive eukaryotic 5S rRNA system intermediate to archaea and the common eukaryotic model system. It may reflect the history of many aspects of the typical eukaryotic 5S rRNA system as it evolved from its archaeal form. Simultaneously, our work implies although the adaptation to a parasitic lifestyle has caused a number of profound changes to G. lamblia, such as a compact genome, mitochondrial relics, and simplified machinery for some metabolic pathways, some very primitive eukaryotic features have been retained by this organism. These have no direct relationship with the parasitic lifestyle but are rather involved in the fundamental functions of a eukaryotic cell. Therefore, we propose that G. lamblia is most likely a combination of eukaryotic primitiveness and secondary parasitic degeneration.
Supporting Information
Table S1.
Putative 5S rRNA genes identified in N. gruberi genome database.
Table S2.
The identified C2H2-type zinc finger proteins in G. lamblia WB genome.
Table S3.
Accession numbers of genes of transcriptional factors and specific subunits of RNA pol III in G. lamblia, T. vaginalis, T. cruzi, and N. gruberi.
Table S4.
Comparisons of the 5S rRNA system between G. lamblia and Encephalitozoon cuniculi.
We thank the Marine Biological Laboratory at Woods Hole for making their Giardia lamblia genome database available to us.
Author Contributions
Conceived and designed the experiments: JFW. Performed the experiments: JMF JS. Analyzed the data: JFW JMF JS DDX. Wrote the paper: JFW JMF JS.
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text:
You've Outlived
Outlived you
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Ernst Dohm
Died when: 63 years 257 days
Star Sign: Gemini
Friedrich Wilhelm Ernst (or Ernest) Dohm (b. Elias Levy Dohm; also known by his pseudonym Karlchen Mießnick; 24 May 1819, Breslau – 5 February 1883, Berlin) was a German editor, actor, and translator. He was Jewish and a convert to Christianity. He married feminist Hedwig Dohm, and had 5 children: 1. * Hans Ernst Dohm (1854–1866) 2. * (Gertrud) Hedwig (Anna) Dohm (1855–1942), married to Jewish scientist Alfred Pringsheim 3. * Ida Marie Elisabeth Dohm (1856–?) 4. * Marie Pauline Adelheid Dohm (1858–?) 5. * Eva Dohm (1860–?) He became a grandfather of the musician Klaus Pringsheim, Sr. and Katharina "Katia" Pringsheim, the wife of Thomas Mann. He was a chief-editor of the Kladderadatsch, a satirical magazine founded in 1848, until 1849.
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