text:
geez....some Jets fans are complete Losers to have to get in his face just because he wants to show support for Mark.
*OR* maybe his POINT was :
In essence, Mark is the QB of the JETS(His T-E-A-M) and will continue to support him because he's the JETS QB. And if(when? hopefully?) Mark is gone, he will support the New guy(whomever that is)....
This is sad..
Hopefully all the obnoxious fans will realize that they're causing a divide among people who are all rooting for the same thing.
Meh, he'll probably enjoy going to games more now. I sat next to him for a season. I hate his schtick, but he is a good guy.
Hey fellas, what's this thread about?
That blows, it really does. The guy's biggest crime was that he was a passionate Jets fan. The Jets P.R. department tried to parlay that into something bigger than it was, and through no fault of his own, it actually worked for a while.
People resented him, why? Jealousy, I suppose. Or just the bald, baseless resentment that is the result of being an ******* in general. "Hey, that guy is having fun interacting with fans -- **** him!" Well, there you go, you bores. The Jets lost the last thing that actually made going to games an enjoyable experience.
Thanks for the memories, Ed.
I bet you're in your glory right now. The sad thing is is that no one knows who you are, and no one will ever know who you are. You're not important. You're not even a blip on the radar. You get your thrills based on how many people click on your threads and respond to you inane posts, but in reality your another middle-management prig in a North Jersey McMansion, probably drowning in debt.
he'll be back next year doing the chant
I said it this summer, Teblow will be te downfall of Tanny and Rex. Why bring this circus to NY is just mind boggling. Tebow is nothing more tan an over-hyped glorified TE who pushes his religious beliefs on others. He is a divisive figure that needs to be shown the door quickly. Just look at some of the Denver players comments today on the CBS pregame show and throughout the season. Enough said.
I wonder if any of his haters will step up and get something going. As much as I love the Jets, the Jets fans are among the lamest in sports when it comes to creating a environment that makes a real home field advantage.
Ed has been a symbol for many years.
That also means that he is a target for opposing fans, individuals wanting to make a name by challenging him and those jealous of his popularity.
There also seems to be an element that feels that if they are miserable, then others must be made miserable as well.
Ed, if you should read this, when I first met you, it was as a 10 year old kid attending his first Jets game. You generously took time to take a picture with me and round up a Jets chant for me. Thank you for your dedication and for becoming a symbol of the best of a Jets fan.
It's unfortunate that the unruly, miserable types finally eliminated a force that represented decency and enthusiasm.


text:
The Horse Forum
- English Riding
- - jumping help?
jumping help? Okay, so technically, I've been jumping for about 5 years now, but I haven't really jumped in a while due to Rio getting lame and buying a green horse.. I'm in dire need of help with my position. I'm leaning too far forward, my heals are UP, my shoulders are in the "defensive position" when I go over the jump(its a crossbar -.-) and I don't know where I should put my hands. I don't think it has anything to do with my leg muscles, I've been doing half seat as practice and I do barn chores 2 nights a week(feeding horses, emptying/filling water buckets/troughs)but everytime I go over the jump... I don't know, I just forget and I go into that defensive mode... Toward the end of my lesson yesterday, I finally got my heels down and my shoulders back.. Do you have any suggestions on how I can get my confidence up? I'm not afraid, he can just walk over the jumps cause they're so low, but I don't know what else is wrong. I've been riding almost every single day since we got him(3 months ago), working on my postion and balance on the ground, but it just goes to crap whenever I get to the jump. My hands are actually not bad, I just need to stop pressing on his neck. Anyways, any suggestions would be lovely! |
Put your sturrips up about 4 holes, tie your reins and have someone lunge you in your two point for awhile. It sounds like your muscles arn't there, I thought mine where, and when we put my sturrips up and got my knee in the right angle, I was not strong at all. |
The only thing I can suggest it try two pointing over ground poles, and work on your position (two point eactly how you want to over a jump) Once you can go over those comftorably in good form, have your trainer put it up to the lowest hole as a cross pole and keep going around like nothing has changed. Good luck! It might take a bit, but once your get relaxed I bet it will all come back to you :-) |
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subset: commoncrawl


text:
October 6, 2010
Escolios a un Texto Implícito: Selección, p. 324
2 comments:
This aphorism is very similar to the last line of "El reaccionario auténtico," an essay published in 1995, one year after Gómez Dávila's death. Here is the last line in Spanish:
El reaccionario no es el soñador nostálgico de pasados abolidos, sino el cazador de sombras sagradas sobre las colinas eternas.
RV Young's translation of this line from the end of "The Authentic Reactionary" is:
"The reactionary is not a nostalgic dreamer of a canceled past, but rather a hunter of sacred shades upon the eternal hills."
This aphorism (#1,994) was originally published in 1986 in the first volume of Nuevos Escolios, while "The Authentic Reactionary" was only published after Gómez Dávila's death. However, I am not sure whether Gómez Dávila wrote the aphorism first and later incorporated it into an essay which he left behind at his death, or whether he wrote the essay first and then used it as material for his aphorisms.


text:
Howl'oween Parade | Blessing of the Animals | Rescues & Adoptions | Lost Dog
Rosie's Dog Beach | Bulldog Beauty Contest | Operation Santa Paws
Off-Leash Dog Parks | Dog Laws in Long Beach
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Donations are being accepted until noon on Fri., Dec. 16 at the specially-marked drop boxes at the following merchants (please call ahead to verify participation). Items will be picked up on the afternoon of Dec. 16 and delivered to shelters on Sat., Dec. 17, 2016. Long Beach, Lakewood & Signal Hill, Calif. 1900 Ocean - Long Beach Calif. (outside LA & OC area)
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text:
Developing a nice terminal for horizontals seems to be the missing piece of the puzzle here. Top of /3/ and /7/ seem out of character to me--they look like graffiti scrawling. And as you say the bottom terminal of /2/ isn't there yet (and that solution when it comes could help you with /Z/ and maybe /E/ and /L/ too). Maybe get your brush pen back out and experiment to find new shape ideas.
Primary tabs
I don't think the B is so bad. Maybe a bit rigid on the left, and you could revise the bottom stem/bowl join.
For 8, what about just a single gap on top?
Oof. I quite like it. What's not working for you?
Better I think. Back to those numbers...
It's a little rough but here's some initial scrawl-and-scans with the brush pen. Are any of these headed in the direction you're thinking?
I disagree about the 8. It is already looking like an S to me.
/8/ conversation fodder:
8: The "B" choice, flipped horizontally.
I suspect that would look really odd. I think A works well.
To virtually all type designers, certainly. But I think not only
would laymen not notice anything funny, they would actually
benefit from the divergence from the "S" (my only reason for
recommending flipping).
But I agree that the "A" choice works, and it might also be
worth closing either the top or bottom of the "B" choice.
Horizontal terminals below. It looks better to have different shaped terminals for the top and bottom of the /E/ instead of having one be the reverse of the other.
The top of the "Z" bugs me a little, but maybe it fits in your scheme.
I like the "2", but it might be better to make the top counter appear
smaller than the bottom (probably by moving the spine up). The "L"
should be narrower than the "E".
Those bottoms are all improvements, especially the /E/.
Top of /2/, like I've noticed in /C/G/c/, has a kind of low droopiness to it that maybe some curve-massaging could lessen.
-sharper angles on top of /7/Z/
-tighter curl at top of /c/C/G/2/
-narrower /L/
All improvements!
I still dont really like that E and alikes, so now I'm thinking maybe you should add that same 'serif' as Z has to the E and alikes.
This will go into bestsellers no matter where you'll upload it. :)
Pardon my disappearance. I've been in ligature-land, building the rest of this thing out. I'll still take a look at those serif issues but in the meantime have a look through the rest of these glyphs. I'm pretty excited about the stylistic sets. Here's some over-the-top, swashy examples:
(thx Minerva)
Love the droplets!
Agreed. This is looking great. These recent embellishments are miles ahead of your first attempts.
I just noticed something: the numerals are too light.
BTW, forget "droplets" - let's call these "squirts". :-)
Hmm. Agreed.
Will you make accented swashes or ligatures or just basic latin?
I can do that. Wasn't sure how much demand there would be for that. Some glyphs probably won't allow for certain accents etc.
Stunning!! Will you create a bold as well or just leave it at this?
Trying to get the color right on these numerals:
That /8/ still bothers me.
In my perfect world, there would be a light and bold face (maybe even an accompanying text face) but am not sure how long that would all take. I can try out some bold glyphs and see if it's worth pursuing.
Keep going with this?
Yes! Have you tried extrapolating to a light?
I'm doing all this by hand, so...not yet. If I had known I would have made light/bold masters and let software do the in-between. I'll play with it. See how it looks.
Nice to see this evolving. What a step from your initial characters!
A peek at the bold under construction:
Looking good. I'd say the same thing I said long ago about the regular: mind balancing the counter sizes, which are again getting too narrow in some cases.
There may be a better solution for the top terminal of /C/G/, which here even more than the regular looks too turned in on itself to my eyes. An additional issue with this shape in the bold is that the /c/ may look too much like an /e/.
Love that /X/x/!
That's no Bold, that's a Demi!
With the Bold, go hog-wild.
Nice! Hrant, this looks like an extrabold to me.
Middle part of /e/ is too thick.
tittles look odd. I think you could extend them more vertically as well.
I don't see any counter-size problems. Perhaps the counters of /uvwxy/ are a bit too large...
Diagonal in /k/ is too light.
bottom of /j/ looks weak.
/EFL/ are too wide.
Bowls of /P/ and /R/ are too small.
Bottom of /U/ needs more weight on the left.
part where the thins of /x/ meet the thick diagonal should have about the same thickness as the part where the middle part of /e/ enters the bowl, which should be thinner in both cases.
/r/ might turn out too wide when set in a word.
OK, OK, it's a Bold (but not more).
But with the Black do go feral porcine.
Ok that's the kick in the ass I need. I don't want to be wishy-washy with the bold. Or black. Or fat, husky, whatever.
Blancmange Obese. That's it.
So there's essentially two types of terminals I'm trying to reconcile here. A "thin" one, like at the top of the /a/ and a bulbous one, like on the top of the /c/. What to do when blowing it up real fat? Any thoughts on some of these options:
I think there's three things you can try: increase
contrast, increase contrast, and increase contrast.
For /a/ go with nr 2.
For /c/ go somewhere between 1 and 2.
Why did you make the joint at the bottom of /a/ thicker? I prefered the previous one.
Hrant. Explain.
I'm just saying the way to make a Bold
alive is just as much through contrast
as it is through weight.
Or were you talking about "feral porcine"?
That was just a joke: hog-wild. Geddit? :-)
That's the Typophile humor we should all by paying money for.
If you extrapolate out the curves from the original weight, you get this:
Looks unorthodox but it seems true to the design. Thoughts?
Put that in a juicer! Yum.
Cool! In all styles you made so far, the bottom right of /c/ needs to be moved to the left a bit. It's falling backwards...
The main UC and LC. No ligatures or numerals just yet.
[bringing those tittles down a bit to fit under the /T/]
/t/ looks short compared to the ascenders.
/T/ looks light.
Some of the tall vertical strokes might be a little stiff looking. Is there a way to soften up those straight (or nearly straight) contours?
/s/ looks a little out of character - maybe too wide, too symmetrical, too smooth?
I think the main problem here is the difference in thickness of the horizontal parts when letters like /c/ and /a/ or /e/ and /s/ are placed next to each other.


text:
06-18-2011, 06:57 PM | #1 (permalink) |
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Posts: 22
What is the Ideal coilover angle in the back?
I see so many builds with so many different set ups. Seams to me the coilovers in the rear would be best and have more room in the range of motion if they are vertical, staight up. This means I would have to cut into the TJ tub well for the hoop. Most people angle the top rearward and do not need to cut the tub. Is that why they do that or is angled actually better?
I just back halfed the frame and it is now moved inboard and the coilovers will be the next step so looking for input. thanks, |
06-21-2011, 06:18 PM | #2 (permalink) | |
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06-21-2011, 06:34 PM | #3 (permalink) |
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Yes it will be a long arm set up with dana 60's and 37" tires set with the belly about 22" off the ground and 12" fox coilovers in the middle of travel and driven on the street occassionally and always from home to the trails even if out of state. I dont plan on rockracing or competive crawling but hard core trails that require the dana 60's with 35 sline and 37" tires. I know there will always be alot of ways to skin a cat and someone will always say I went about it the wrong way or to much or to little. It this enough information to help me decide if I should lean my shocks back in the tub and no cutting or go straight up and cut out the wheel well?
06-21-2011, 08:46 PM | #4 (permalink) | |
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Well.. 12" with even up/down travel means a pretty short hoop. I'd probably be included to lean them to the rear. With only 12" of travel it really leaves a lot open.
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06-22-2011, 08:27 AM | #5 (permalink) |
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The tops of the front CO's should lean back and in.
The tops of the rear CO's should lean forward and in. The reason you see the bolt on CO set ups all leaning the rear shock back is for packaging. The type of person who wants a kit (and some custom rigs too), also doesn't want to cut into their body so the manufactures make a product to fit the bill. There is a bunch of information on the subject if you just search. I didn't search as well as I should have before I built mine and had to rebuild all the mounts. I originally built mine perpendicular to the axle at rest (which would have been correct if I was just making shock mounts, but for CO's its different) but had to redo them because it was immediately obvious there would be a stability issue. The reason they go back in the front and forward in the back is because of the arc of the control arms. And the reason they all angle in towards the center of the vehicle is because of progressive and regressive spring rates (makes it feel tippy if you don't). Ben |
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06-22-2011, 11:22 AM | #7 (permalink) |
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In my experience the ideal angle is between 10-15 degrees forward and the same angle in... That allows for maximum stability on sidehills and uphills... If you have front coilovers the should have the same angle in and toward the rear. That would give additional sidehill stability and give stability coming downhill. Usually any more angle than 15 degrees in any direction means that you will need special valving.
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06-22-2011, 11:28 AM | #8 (permalink) |
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Inward angle should be set up so that when fully flexed, the stuffed said maintains the shock at 90* to the axle.
And the lean front to rear is going to depend on who you talk to. If you angle them away from the direction of the links, the spring rate stays more linear, instead of changing as suspension moves. That's why spring rate calculators ask you the angle, it needs a differant spring rate.
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"He was quite a lad, to put it mildly," maintains Christopher Lee about Ian Fleming, who "wrote the spy thriller to end all spy thrillers".
James Bond gets yet more publicity here, but this proficient documentary, which blends interviews with Bond footage, thankfully doesn't shy away from the squabbles (mainly between Connery and the producers) that have plagued the franchise. Plus, the short segment with George Lazenby, a male model who had never acted, explaining how he bagged the role, is fascinating.
Reuse content


text:
Definition of Sperse
1. v. t. To disperse.
Definition of Sperse
1. to disperse [v SPERSED, SPERST or SPERSED, SPERSING, SPERSES] - See also: disperse
Sperse Pictures
Click the following link to bring up a new window with an automated collection of images related to the term: Sperse Images
Lexicographical Neighbors of Sperse
Literary usage of Sperse
Below you will find example usage of this term as found in modern and/or classical literature:
1. A Glossary: Or, Collection of Words, Phrases, Names, and Allusions to by Robert Nares, James Orchard Halliwell-Phillipps, Thomas Wright (1901)
"To SPARSE, or sperse. To scatter; from the Latin. ... See sperse. Spent. rq,iis».
SPA RVER, *. The canopy or tester of a bed ; evidently so, ..."
2. The Rhyming Dictionary of the English Language: In which the Whole Language by John Walker, John Longmuir (1902)
"To sperse To disperse, va To a-sparse' To slander; to censure, va To dic-perse' To
... drive away; scatter, va To in-ttr-sperse' To mingle here and there, ..."


text:
MTV's Multiplayer speculated and has now confirmed that Microsoft is currently hiring new team members for Peter Jackson's mysterious Halo-related project. The speculation arose when Microsoft posted several new job listings looking for people to "help develop a new experience in the Halo universe" alongside an "external luminary." So, Halo and a luminary. Sounds like Peter's project to us.
While at first it seemed strange that Microsoft would only now be hiring for the project, Microsoft followed up with MTV to clear things up. The company told MTV, "Microsoft Game Studios has been hard at work collaborating with Peter Jackson and Bungie on the development of [a Peter Jackson-helmed Halo project]. At this time, it's necessary to grow the internal team to keep pace with development. Bungie will continue to remain involved in multiple aspects of the Halo Universe with MGS." So, it turns out that the project's development team is merely expanding -- as opposed to just now being formed -- which would indicate the project has become more expansive as well.
Let's hope we can hear some news about the actual game sometime soon. Seriously, we're starving for info.
[Via Joystiq]


text: Dragon Quest X Coming To WiiMichael McWhertor12/10/08 12:40amFiled to: Square EnixWiiNintendoDragon quest xDragon QuestTopNews141EditPromoteShare to KinjaToggle Conversation toolsGo to permalink Yuji Horii, creator of the Dragon Quest series, revealed at Square Enix's press conference in Tokyo today that the next entry in the role-playing game series, Dragon Quest X, is in development for the Wii. Advertisement After announcing the final release date for Dragon Quest IX, which will be released for the Nintendo DS in Japan on March 28 for 5980 yen, Horii confirmed that the series would be returning to consoles for its tenth entry, according to Japanese game site Gigazine (via NeoGAF). Square Enix released a spin-off, Dragon Quest Swords: The Masked Queen and the Tower of Mirrors, for the Wii in 2007, but DQ X will be the first release in the core series for the Wii platform.The revelation comes two years after the announcement that DQ IX would be released on the Nintendo DS after two entries on the PlayStation 2. Reply14 repliesLeave a reply


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Locally owned, operated, and grown, Horseshoe Point Honey LLC is a small, sustainable, beekeeping operation in the North East section of Suffolk, VA. Dedicated to raising bees in a natural & healthy way, we offer 100% pure raw honey—just how the bees made it. Never heated or processed, no chemicals used, and only gently screened so that the natural pollen is maintained to aid allergy sufferers.
Unlike 'store bought' honey, which is normally heated (pasteurized) and ultra-fine filtered, our honey is unprocessed, raw, and naturally healthy. Many allergy sufferers attest that consuming local honey reduces their seasonal allergies because of the small amounts of local pollen maintained in the honey. Unprocessed honey also contains small amounts of a wide array of vitamins, minerals & amino acids, plus antioxidants. Honey is nature's energy booster, providing a concentrated energy source that helps prevent fatigue. Also, latest research proves that honey is a better remedy than any over-the-counter cold remedies for sore throats and coughs.
There's an important difference between what you get at the supermarket and what you get from a trusted, local beekeeper. When honey is processed commercially, it is often finely filtered and heated. Filtering removes most pollen particles and the heat can change the color, taste and, most importantly, destroys the vitamins and most of the natural health benefits. Additives have also been discovered in commercially processed honey.
Local honey taken for allergy relief is defined as within a 50 mile radius of your location because vegetation and, thus pollens, do not vary much within 50 miles. Local honey contains the same offending pollens that seasonal allergy sufferers need to address. We use the coarsest mesh filters possible when straining the honey to ensure the beneficial pollen is retained in the honey. All of the Hampton Roads Area of Virginia is within 50 miles of our bee hives. Norfolk is only 15 miles away.
Horseshoe Point Honey LLC is state certified by the VA. Dept. of Agriculture to process (extract and bottle) our honey. We use no chemicals or pesticides and are careful to ensure our bees are not exposed to pesticides.
Horseshoe Point stands behind its honey and believes you will classify it among the best honey you can find.


text:
Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications. | |
Jump to Full Text | |
MedLine Citation:
PMID: 21548984 Owner: NLM Status: MEDLINE |
BACKGROUND: Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species. RESULTS: Intron-F's (inserted at L798 position) were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE. CONCLUSION: The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species. |
Kayoko Takizawa; Toko Hashizume; Katsuhiko Kamei |
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Publication Detail:
Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2011-05-08 |
Journal Detail:
Title: BMC microbiology Volume: 11 ISSN: 1471-2180 ISO Abbreviation: BMC Microbiol. Publication Date: 2011 |
Date Detail:
Created Date: 2011-06-13 Completed Date: 2011-09-08 Revised Date: 2013-06-30 |
Medline Journal Info:
Nlm Unique ID: 100966981 Medline TA: BMC Microbiol Country: England |
Other Details:
Languages: eng Pagination: 94 Citation Subset: IM |
Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan. email@example.com |
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MeSH Terms | |
DNA Primers
genetics Environmental Microbiology Genotype Humans Introns* Molecular Sequence Data Mycological Typing Techniques Mycoses / microbiology Nucleic Acid Conformation* Phialophora / genetics*, isolation & purification Phylogeny* Polymerase Chain Reaction / methods RNA, Ribosomal, 28S / genetics* Sequence Analysis, DNA Sequence Homology, Nucleic Acid |
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Reg. No./Substance:
0/DNA Primers; 0/RNA, Ribosomal, 28S |
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Full Text | |
Journal Information Journal ID (nlm-ta): BMC Microbiol ISSN: 1471-2180 Publisher: BioMed Central |
Article Information Download PDF Copyright ©2011 Takizawa et al; licensee BioMed Central Ltd. open-access: Received Day: 30 Month: 11 Year: 2010 Accepted Day: 8 Month: 5 Year: 2011 collection publication date: Year: 2011 Electronic publication date: Day: 8 Month: 5 Year: 2011 Volume: 11First Page: 94 Last Page: 94 ID: 3112068 Publisher Id: 1471-2180-11-94 PubMed Id: 21548984 DOI: 10.1186/1471-2180-11-94 |
Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications | |
Kayoko Takizawa1 | Email: firstname.lastname@example.com |
Toko Hashizume2 | Email: email@example.com |
Katsuhiko Kamei1 | Email: email@example.com |
1Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8673, Japan |
2Advanced Engineering Services Co. Ltd., Tsukuba Mitsui Building, 1-6-1 Takezono, Tsukuba, Ibaraki, 305-0032, Japan |
The type species Phialophora verrucosa was described by Medlar in 1915 [1] when he isolated the fungus from a human skin disease. The species is ubiquitous and cosmopolitan, and are important plant saprobes as well as human pathogens. Identification is based on conidial ontogeny and molecular systematics. Few studies involving molecular genotyping techniques have been reported for P. verrucosa. A study analyzed restriction fragment length polymorphisms (RFLP) of mitochondrial DNA to determine genetic variations and phylogenetic relationships among P. verrucosa strains [2].
Different molecular typing tools, such as random amplification of polymorphic DNA (RAPD), RFLP, pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST), have been developed to provide a better understanding of the molecular epidemiology of fungal pathogens, e.g., Candida albicans [3-5] and Aspergillus fumigatus [6,7] and medically important filamentous fungi [8]. However, although the majority of the reported group 1 intron sequences have been found in a wide range of fungi (Comparative RNA Web [CRW] site: http://www.rna.ccbb.utexas.edu/[9], few studies about sequence and structure variation, distribution and phylogenetic relationships of introns from a single species have been performed in detail. We focused on group 1 introns within 28S rDNA from P. verrucosa to evaluate the prevalence of intron polymorphism at the strain level.
As the first step to determine intron sequence divergence, sequences of 28S rDNA of five representative strains of P. verrucosa were analyzed to find insertions. Based on these five sequences, site-specific primers were designed for use in PCR to detect insertions on other P. verrucosa and P. americana strains studied, in order to investigate incidence and distribution of insertions. Moreover, to characterize the insertions, we analyzed the phylogeny of the introns found in this study and predicted their secondary structures.
Since the sequence information of the 28S region was not available in public databases, we sequenced the 28S, including ITS, regions of five representative strains (Table 1) of P. verrucosa. Alignment of the five P. verrucosa rDNA sequences revealed the nucleotide sizes of 18S, ITS and 28S and the location of intron-F and G insertions (see Table 2 and Additional file 1). It was found that the sequences were composed of 29 nucleotides of partial 18S, 534-535 nucleotides of ITS and from 3349 to 4133 nucleotides of 28S regions. These genomic sequences were deposited at DDBJ and accession numbers are listed in Table 2. Twenty-five and two nucleotide substitutions were found within the ITS and D1D2 regions, respectively. The two bp substitutions in D1D2 were located at 1036 and 1042 nucleotide positions in the 28S region. These polymorphisms were confirmed in the five strains of P. verrucosa strains except for the two insertions in intron-F and G at 924 and 2239 positions of the 28S, respectively. None of the insertions were found in the Yao strain. BLAST comparisons showed with relatively high homology values that the sequences of both insertions were individually homogeneous to group 1 introns in the database. The positions of insertion for intron-F and G correspond to nucleotide positions 798 and 1921 of Escherichia coli 23S nucleotide sequence accession number J01695 [10]. In addition, although these five strains of P. verrucosa did not possess intron-H, this region of PV28 strain was amplified with the site-specific primer pair (in Table 3) designed from the sequences obtained from another experiment (data not shown) and a 403-nucleotide insert representing intron-H was sequenced. The insertion positions of the intron-H correspond to nucleotide positions 2563 of E. coli 23S nucleotide sequence.
We amplified intron insertion regions using site-specific primer pairs we have designed for intron-F (inF-F and inF-R), intron-G (inG-F and inG-R) and intron-H (L2563F and L2563R), within the 28S region (Table 3). These primer pairs were used to screen and detect PCR amplicons for insertion regions within 34 P. verrucosa and seven P. americana strains. Amplicons were eluted in agarose gel to gain information regarding the intron insertions. No-insertion amplicons for intron-F and intron-G primers were in the size 142 and185 bps, respectively. When insertions were present, intron-F primer pair yielded amplicons in the size range from 531 to 533 bps, and intron-Gs in the size 575 or 578 bps. Moreover, amplicons of about 643 bps for intron-Hs were also eluted. It was revealed that there were 30 intron-F's, four intron-G's and six intron-H's within P. verrucosa and only two intron-Fs within P. americana as shown in Table 1. There was some correlation between intron distribution of P. verrucosa and geographic location, i.e., intron-Fs were found to have prevalence of 88% in P. verrucosa and intron-Hs were found specifically in the South American Continent. No introns were found except for two intron-Fs in P. americana. In addition, the agarose gel profiles allowed us to characterize genotypes and distribution frequencies of insertions from P. verrucosa including no-insertion as shown in Table 1. It was found that occurrence of genotypes F, FG, FH, FGH and N were at 64, 6, 12, 6 and 12%, respectively.
RT-PCR was carried out to identify the property of these insertions, namely, whether they are introns or unusual extensions incorporated into mature rRNA. Four representative strains were selected among the 41 strains surveyed. And it was found that two strains (PV1 and PV3) had two introns individually, while the other two strains (PV2 and PV41) had only one intron as shown in Figure 1. Insertions of strain PV1 and PV3 were eluted at 142 bps on lane 2 and 3 with intron-F primer pair, and 185 bps on lane 4 and 5 with intron-G primer pair, respectively. PV2 and PV41 exhibited 142 bps amplicons with intron-F primer pair as shown on lane 15 and 16, respectively. An intron-lacking Yao strain gave 142 and 192 bps amplicons with intron-F and G primer pairs on lane 10 and 11, respectively. The other lanes; namely, 6, 7, 8, 9, 13 and 14 show PCR products of genomic DNA as templates and lane 12 is negative control. These shows that all the insertions were excised after cDNA was transcribed, indicating that they were, as predicted, actively spliced introns. These results point to the possibility that these insertions are group 1 introns.
Moreover, we analyzed sequences of the spliced introns to confirm the boundaries of exon and intron sequences. The last nucleotide of the upstream exon was confirmed to be a T (U in RNA) and the last nucleotide of the intron was a G, consistent with group 1 introns [11,12].
Sequences of intron-F and G of ten P. verrucosa strains were sequenced and it was found that DNA sequence polymorphisms exist among the two introns, i.e., the intron-Fs ranged in the size from 389 to 391 bps and the four intron-Gs from 389 to 393 bps shown in Table 2. There were 24 nucleotide substitutions and two deletions/insertions (TH9 strain) within intron-F. There were five nucleotide substitutions among intron-Gs from PV1, PV33 and PV34, unlike 36 substitutions between PV1 and PV3. In addition, Blast search analyses and alignment lead us to believe that intron-Fs and Gs from 14 introns belong to subgroup IC1 of group 1 intron.
Fourteen introns from 12 representative strains of P. verrucosa including Tetrahymena thermophila as out-group were aligned and used for phylogenetic analyses. Neighbor-joining (NJ) and Maximum Parsimony (MP) trees based on the alignment of these intron sequences are shown in Figure 2. The data set consisted of 466 characters, of which 156 were removed from the MP analysis due to ambiguous alignment. Of the remaining 310 characters, 201 were variable and 129 were phylogenetically informative for parsimony analysis. Three major distinct and well-supported clades that had homologous topology were obtained from both phylogenetic analysis methods showing that all the introns analyzed were undergoing a similar rate of evolution. The first clade [I] (87% BS support in NJ, 81% in MP) consisted of six strains having intron-F including 3 clinical isolates, the second clade [II] (57% BS in NJ and 77% in MP) consisted of 4 strains having intron-F, and the third clade [III] (100% BS in both trees) consisted of four G introns. All the introns clustered in clades [I] and [II] are inserted at the same position L798 those in clade [III] at the same position L1921. Introns inserted at the same positions belong to the same clusters and are considered to be the same subgroups. We observed that two Venezuelan strains in clade [II] (TH31 and TH35) included the remnants of homing endonuclease (HE) gene sequences, which were found to invade into IC1 introns of the 18S regions in another study (unpublished data).
Alignment of the core regions consisting of highly conserved sequences of the elements of P, Q, R and S and the pairing segment P3 and the nucleotide sequences, in particular, the last two nucleotides GC of the Q element and the first and second GU nucleotides of the R element [12] (Additional file 2) showed that the introns belong to group IC1. All core region sequences of intron-Fs were found to be identical. Two sequences of core regions termed as intron-G (PV3) and intron-G (PV1, PV33, PV34) were obtained and added to the NJ analysis in Figure 3. The NJ tree was constructed based on the alignment of these core regions consisting of three representative sequences of P. verrucosa and IC1 of 21 taxa drawn from database using IE intron from Neoscytalidium dimidiatum as out-group. The phylogeny of intron-F and G formed separate clades as shown in Figure 3, and indicated that both introns were likely acquired independently. Indeed, all intron-Fs were found to be closely related to Myriosclerotinia ciborium and Sclerotinia tetraspora introns which are located at L798. Two sequences of intron-G located at L1921 were grouped together with 85% BS value and found to be on the neighboring clade with Cordyceps prolifica intron located at L1921. The phylogenetic tree suggests that both introns may be inserted prior to the divergence of the species formerly belonging to clade [IV] and [V]. Collectively, this tree displays that all introns of P. verrucosa generated by the core regions are members of subgroup IC1s.
The predicted secondary structure of the intron-F and G were constructed as follows. The conserved P, Q, R and S regions of intron-F (L798) from PV1 were initially aligned with the same regions from other taxa, and then regions of P1 through P10 were constructed and added on the basis of the secondary structure model as shown in Figure 4[A][13]. The P10 region was formed using the internal guide sequence (IGS). The catalytic core was defined by a set of structurally conserved elements, including elements P3 to P8. A G-C pair within P7, i.e. G391-C277 of intron-F was assumed to be G-binding positions [14]. Extended P5 and P9 stems were displayed in the putative structure of intron-F from PV1. Nine intron-Fs from nine strains (PV2, 3, 28, 33, 34 and 41 and TH9, 31 and 35) of P. verrucosa were predicted to be the same structures as the putative structure of intron-F derived from PV1 drawn in Figure 4[A], alternatively, shown in Additional file 3. These nucleotide variations among intron-F were observed mainly in the loop and at four positions where one nucleotide of P5a, two of P5.1a and one of P5.2 stem were positioned. The base pairs GU and CG within P6 were formed in the core region of intron-F [12]. The nucleotides A71, A72, U73 were located in segments J3/4 of PV1 intron-F [15-18]. These predictions of secondary structure revealed that all intron-Fs were IC1 group 1 introns.
The structure of intron-G (L1921) from PV1 was drawn just as was done for intron-Fs (Figure 4[B]). A G-C pair within P7, i.e. G390-C360, was assumed to be the G-binding positions. The GU-CG pair of P6 and the AAU in J3/4 was the same as in the intron-F core region of PV1. This putative intron-G exhibited expanded regions of P1 and P5. The three intron-Gs of PV1, PV33 and PV34 were found to be similar among the three strains. Different features were found in PV3 as shown in Figure 4[C] wherein the sequence of PV3 differed in P1 region among four trains; namely, short stems in P1b and P1c and small bulge loops of L1 and L1a (Additional file 4). Moreover, PV3 added P2.0 and P8c, although the other intron-Gs did not. Prediction structures in the remaining two introns of PV33 and PV34 are not shown. Nevertheless, all subgroups of intron-G were also identified as IC1, based on comparison of tertiary structures across segments P3-7 of the four strains.
In conclusion, we have identified that the ten intron-Fs and four intron-Gs of P. verrucosa belong to IC1 group 1 introns.
Loss of P5abcd domain in derived S788 introns was correlated with inability to self-splice in vitro in a previous report [19]. Accordingly, we have not confirmed insertion positions of intron-H by RT-PCR. However, we examined PV-28 strain as the representative strain of intron-H by analyzing the sequence alignment of the core region of subgroup IE from other organisms in the database. Moreover, we predicted the secondary structure of this intron-H as shown in Figure 5. The secondary structure modeling revealed distinctive IE sequences, namely, the non-expanding P5 and the expanding P9 regions.
To date, although a variety of introns from eukaryotes have been described in the rRNA gene loci of fungi [9], few introns in Phialophora species have been reported. An unusually small group 1 intron of 67 bps from the nuclear 18S rDNA has been described in a splicing study of Capronia semiimmersa, a teleomorph of P. americana which is known to be similar to P. verrucosa [20-22]. These small introns contain only P1, P7 and P10 elements, because most of the core regions common in almost all other group 1 introns are missing. Four intron sequences have been reported or registered in dematiaceous fungi; namely, 283 bps within the small subunit (SSU) rDNA from Cadophora gregata f. sp. adzukicola [23], 339 bps within SSU from Cadophora finlandica (accession number: AF486119), 456 bps within the large subunit (LSU) rDNA from C. semiimmersa [24] and 397 bps within LSU from Cladophialophora carrionii [24]. These introns have not been subjected to secondary structure analysis. Therefore, we aimed to identify the introns that we found in this study and to investigate the prevalence and phylogenetic relationships of 28S group 1 intron at the intra-species level.
The intron-F, G and H in the 28S rDNA of both species were found to belong to two subgroups, IC1 and IE, of group 1 intron. IC1 at L798 is the most common insertion position as shown in Table 1 and in the CRW website, and insertions at L1921 and L2563 were found comparatively in the database. The loss of most of P5 in the secondary structure of intron-H is believed to be a relatively recent evolutionary event [19]. The three insertions possessed all the ten elements (P1-P10) common in group 1 introns. Enzymatic core regions are especially well conserved in primary and secondary structures, as described in previous reports [12,25], suggesting that they were derived from a common origin. Peripheral elements of the core have various forms and these variations have been used to subdivide introns into five major subgroups [17,26]. In this study, the phylogeny obtained in Figure 2 and 3 showed that all IC1 introns inserted into P. verrucosa have been surviving with base substitution/insertion/deletion, especially among peripheral elements as a consequence of some events after the individual insertion of IC1 at L798 and L1921, and may have spread by homing (e.g., [27-29]) or reverse splicing [30-32].
Comparisons of intron-F and G indicate comparative high sequence divergence within P. verrucosa wherein the sequence similarity among intron-F's was 94%, and 99% among intron-G's with the exception of PV3 and 90% among all the four intron-G's. The phylogenetic relationships among both introns were very similar to each other (Figure 2). These results suggest that they have diverged from a common origin.
Thirty isolates of P. verrucosa and two isolates of P. americana possessed intron-F, G and H either individually or as a combination of these introns. Genotypes based on the combinations of presence or absence of introns, type and position of insertions were established to discriminate among the isolates surveyed. As a result, five genotypes; namely, F, FG, FH, FGH and N were identified, as shown in Table 1. Type-F was isolated in all the countries where the strains used in the study. Intron distribution was found to have some correlation with geographic location, albeit the number of isolates used was small. For example, most of the Chinese isolates except for Yao-strain had only type-F. Isolates from South American continent had slight tendency to have an intron-H, wherein they were either type-FH or FGH. Intron-G's occurred as type-FG in the clinical isolates of Japan and China and as two type-FGH's in soil isolates of Brazil. In addition, according to an interpretation from a different viewpoint, insight into possible correlation of geographic origin among introns from P. verrucosa strains have emerged from these insertion position results, namely, the spread of L798 among a large number of P. verrucosa isolates and the existence of L1921 and L2563 that coexist with the other intron insertions among the species and strains that have lost introns. L1921 positions are only seen in two clinical isolates from Japan and China and two isolates from Brazilian soil. The L2563 position seems to be specific to the South American continent in six environmental isolates. The possible correlation tendencies shown in our results have also been reported in previous studies described below. For example, group 1 intron CgSSU was found in the SSU rDNA of deuteromycetes mycorrhizal fungus Cenococcum geophilum, and the intron-positive isolates occurred mainly in North America and Europe and negative isolates in Western, Midwestern and Southern North America [33]. In other studies on intron distribution from a single species, four different group 1 intron combinations within LSU rDNA from entomopathogenic hyphomycete Beauveria bassiana were divided into 13 genotypes to investigate distribution frequencies in the population and it was found that there was a tenuous correlation with geographic origin or insect host species [34]. Moreover, M. Márquez, et al. have found three intron insertion positions within LSU rDNA and established seven genotypes among 26 biocontrol isolates for entomopathogenic anamorphic Metarhizum anisopliae [35].
Meanwhile, we found that five isolates of P. americana had no introns, even though two isolates were detected as type-F. Intron-loss strains might have been lost from taxa possessing intron-F in the common ancestor of the species during its evolution [36]. Further, the lateral transfers appear to have been rare events in P. americana. The fact that intron-F was found in almost all isolates of P. verrucosa, it is believed that intron-F may be specific to P. verrucosa. To confirm this hypothesis, more isolates are needed in the survey and the relationships of the clinical background of the individual patients and the ecological niches of saprobic isolates must be investigated. Further analysis of genotypes within the complete nuclear rDNA gene must be done and the presence of HE gene sequences must be analyzed since they provide key information on intron phylogeny and origin. This study is a first step in the study of introns in P. verrucosa and P. americana.
The three insertions within 28S rDNA of clinical and environmental isolates of P. verrucosa and P. americana allowed us to characterize them into five genotypes using agarose gel electrophoresis patterns. The two insertions, namely, intron-F and G, were characterized as subgroup IC1 by subjecting them to RT-PCR, secondary structure and phylogenetic analysis to determine whether they are true introns, to characterize subgroup and to infer evolutionary relationships, respectively. Another insertion, intron-H, was characterized as an IE intron using BLAST search and by prediction of secondary structure. Furthermore, we also developed a system to classify genotypes based on the presence and distribution of group 1 introns and the distributions as DNA polymorphism among the two species.
We studied 34 P. verrucosa strains including of five clinical isolates as shown in Table 1. Seven P. americana strains including of three clinical isolates were used as allied species. All the isolates were preserved by using L-drying method and were sub-cultured on potato dextrose ager (Difco) slant before extraction of genomic DNA. For an extraction of total RNA, liquid cultivation was performed in 50-ml Erlenmyer flask containing 20 ml of potato dextrose medium at 30°C for seven days on a rotary shaker at 120 rpm.
DNA extraction was performed using an InstaGene Matrix extraction kit (BioRad, Hercules, CA, USA) according to the manufacturer's instructions with minor revisions. Particularly, cells were ground with micro pestle before incubation at 56°C. The extracted DNA was then diluted 1:10 and used as template DNA for PCR amplification.
Total RNA was extracted by using the Nucleic Acid Purification Kit MagExtractor (TM -RNA- TOYOBO, Osaka, Japan). The following procedures were done before carrying out the manufacturer's instructions. Approximately 20 mg (wet weight) of mycelia were washed with water and then rinsed with Schizosaccharomyces pombe spheroplast buffer (20 mM citrate-phosphate buffer (pH 5.6), 50 mM EDTA and 0.9 M sorbitol). This was followed by addition of 100 μl of buffer plus 20 units of Lyticase (L-5263; SIGMA, MO, USA) and 0.01 units of Chitinase (C-7809; SIGMA, MO, USA). The suspension was mixed by vortexing, incubated at 37°C for 30 min and added with 700 μl of a lysis-adsorption solution including 1% 2-mercaptoethanol and pricked with a pipette tip until the solution became viscous. The extracted RNA was treated with RNase-Free DNase Set (QIAGEN). Approximately more than 20 ng/μl RNA was obtained.
A primer walking method was performed to obtain the sequences of the entire 28S rDNA region including ITS. PCR Master Mix (Promega, Madison, WI, USA) and TaKaRa LA Taq (TAKARA Bio Inc, Sigma, Japan) were used depending on the amplification sizes. The PCR conditions for PCR Master Mix consisted of denaturation for 4 min at 95°C, followed by 30 amplification cycles of denaturation at 94°C for 1 min, annealing at primer-dependent temperatures based on Tm values for 1 min and extension at 72°C for 1.5 min, and then 1 cycle of 5 min at 72°C. For TaKaRa LA Taq consisted of denaturation for 1 min at 94°C, followed by 30 cycles of denaturation at 98°C for 5 sec, annealing at primer-dependent temperatures for 30 sec and extension at 72°C for 2 min, and then 1 cycle of 72°C for 10 min. PCR products were purified with SAP-IT (USB Corporation, Cleveland, OH, USA) and then sequenced with primers listed in Table 2 and the BigDye Terminator v3 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an ABI Prism 3130 × l Sequencer (Applied Biosystems, Hitachi). The nucleotide sequences were determined from both strands. To determine base substitutions and intron insertion positions, sequences were aligned by using the alignment function of GENETYX ver. 9.1.1 (GENETYX COOPERATION, Tokyo, Japan).
The extracted DNA was used as template DNA for the amplification of the insertion regions (intron-F, G and H). PCR was performed individually using PCR Master Mix and the primer pair inF-F and inF-R for intron-F and inG-F and inG-R for intron-G which we newly designed. Primer pair L2563F and L2563R for intron-H was designed based on sequences of exon and group 1 intron on CRW website, because the intron was not inserted in the five representative strains used. PCR conditions were the same as described above and the resulting DNA fragments were resolved by electrophoresis on a 2% agarose gel (NuSieve® 3:1 Agarose, TAKARA Bio Inc, Sigma, Japan) in Tris-borate-EDTA buffer. Presence or absence of individual intron was listed as positive/negative in Table 1. In addition, the strains were categorized into five intron types; namely, F, FG, FH, FGH and N on the basis of the intron insertions.
The RT-PCR from total RNA was performed using a SuperScript ™ III One Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, CA, USA) according to the manufacturer's instructions. Ten microliters of RNA was used for a 50 μl reaction mixture consisting of 25 μl of ReactionMix, 2 μl of SuperScript™ III RT/Platinum Taq Mix, 1 ul of each primer inF-F (10 μM) and inF-R (10 μM) for RT-PCR of intron-F and 11 μl of autoclaved distilled water. For RT-PCR of intron-G, primer pair inG-F and inG-R was used. RT-PCR was carried out in the following conditions: cDNA synthesis at 55°C for 30 min, denaturation at 94°C for 2 min, and PCR amplification at 40 cycles of 94°C for 15 sec, 55°C for 30 sec and 68°C for 1.5 min and final extension at 68°C for 5 min. Amplification products were eluted in 3.5% polyacrylamide gel in tris-acetate-EDTA buffer on an electrophoresis run condition of 100 V for 30 min and followed by 75 V for 25 min, together with genomic DNAs amplified with the same primer pairs as control (shown in Figure 1).
The RT-PCR products were purified with the SUPREC-PCR (TAKARA Bio Inc, Sigma, Japan) and ligated into the pGEM-T Easy Vector System (Promega, Madison, WI, USA). Plasmids were transformed into E. coli competent cells (ECOS TM Competent E. coli, JM109, NIPPON GENE Co., LTD., Japan). Transconjugants were selected on LB agar plates containing 50 μg/ml ampicilin and 40 μg/ml of 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-Gal). The presence of the expected insert was confirmed by PCR and agarose gel electrophoresis. The inserts were sequenced with T7 (5'-TAATACGACTCACTATAGGG-3') and M13 reverse primers (5'-AGGAAACAGCTATGACCATGA-3').
Nucleotide sequences were aligned using the BioEdit program version 188.8.131.52 [37]. For phylogenetic analysis, alignment gaps were treated as missing data and ambiguous positions were excluded from the analysis. NJ analysis [38] as distance matrix method and MP analysis as character state method were carried out using PAUP 4.0b10 [39]. For NJ analysis, the distances between sequences were calculated using Kimura's two-parameter model [40]. MP analysis was undertaken with the heuristic search option using the tree-bisection-reconstruction (TBR) algorithm with 1000 random sequence additions to find the global optimum tree. All positions were treated as unordered and unweighted. The maximum tree number was set at 104. To estimate clade support, the bootstrap procedure of Felsenstein [41] was employed with 1000 replicates in both MP and NJ analyses. Bootstrap (BS) values higher than 50% are indicated.
For the comparison with highly conserved sequences of subgroup IC1 from 20 taxa, sequences of elements of P, Q, R and S and the pairing segment P3 were obtained from DDBJ database (accession numbers shown after sample name in Figure 3). These regions do not include IGS, because the sequences in the upstream region of intron insertion positions do not share a common IGS [42]. The NJ tree was constructed after alignment of all the sequences, which ranged from 57 to 60 bps (Additional File 2). Insertion positions are shown after the sample ID or accession number. The insertion position numbering of the taxa refers to the 23S nucleotide sequence of E. coli [10] except for the Exophiala calicioides which is based on the 18S of Saccharomyces cerevisiae. Taxonomic affiliation was indicated by letters in parentheses; namely, [A], Fungi/Ascomycota; [Ac], Acanthamoebidae; [C], Chlorophyta; [H], Heterolobosea; [M], Mycetozoa and [R], Rhodophyta.
The secondary structures are proposed from modeling by Michel et al. [14,26,43] and computational analysis was done using the Mfold web server available at http://mfold.rna.albany.edu/[44] and GENETYX Ver.9 software, with manual adjustments. The pairing segments of P1-P10 locations are indicated in Figure 4 and 5. Moreover, the model was manually optimized based on previous studies of group 1 introns [17,45-47].
KT: conceived the study, designed the experimental plan, performed the experiments, wrote and revised the manuscript. TH: performed the experiments. KK: participated in the coordination of the study, helped draft and revise the manuscript. All authors read and approved the final manuscript.
Schematic representation of the large ribosomal subunit 28S gene. The hatched and dotted boxes correspond to the group 1 intron of P. verrucosa inserted at positions 798, 1921 and 2563 relative to the 23S rDNA of the E. coli J01965 sequence. The numbering in the parentheses is relative to the ITS and 28S rDNA sequence of P. verrucosa.
Click here for additional data file (1471-2180-11-94-S1.PDF)
Additional file 2
Partial alignment of IC1 introns of P. verrucosa and selected introns from the database. Highly conserved sequences of the elements of P, Q, R and S and the pairing segment P3 are also shown. Intron insertion positions relative to E. coli are given after the sample ID or taxon name. * indicates the insertion position relative to the 18S rDNA of the S. cerevisiae sequence. Letters in parentheses indicate taxonomic affiliation: [A], Fungi/Ascomycota; [Ac], Acanthamoebidae; [C], Chlorophyta; [H], Heterolobosea; [M], Mycetozoa; [R], Rhodophyta.
Click here for additional data file (1471-2180-11-94-S2.PDF)
Additional file 3
Alignment of intron-F used for the phylogenetic analysis and the modeling of secondary structure. The gaps were marked with dashes. The highly conserved (ribozymatic core) regions of the P, Q, R and S were marked with dotted lines. Boxed nucleotides participate in the pairing segments of P1-P10 of the secondary structure model.
Click here for additional data file (1471-2180-11-94-S3.PDF)
Additional file 4
Alignment of intron-G used for the phylogenetic analysis and the modeling of secondary structure. The gaps were marked with dashes. The highly conserved (ribozymatic core) regions of the P, Q, R and S were marked with dotted lines. Boxed nucleotides participate in the pairing segments of P1-P10 of the secondary structure model.
Click here for additional data file (1471-2180-11-94-S4.PDF)
This study was supported in part by the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Thirty-four strains of P. verrucosa and seven strains of P. americana examined in this study
Strain | Isolation source | Locality | Insertion | Genotype | Sample ID | ||
Intron-F | Intron-G | Intron-H | |||||
IFM 4928 | Human | Japan | + | + | - | FG | PV1 |
IFM 5089 | Human | Japan | + | - | - | F | PV2 |
IFM 51934 | Human | China | - | - | - | N | Yao |
IFM 41779 | Human | China | + | + | - | FG | PV3 |
IFM 41750 | Bark | China | + | - | - | F | TH9 |
IFM41710 | Corn | China | + | - | na | F (na) | |
IFM 41780 | Human | China | + | - | - | F | |
IFM 41721 | Rotten wood | China | + | - | - | F | |
IFM 41724 | Rotten wood | China | + | - | - | F | |
IFM 41725 | Straw | China | + | - | - | F | |
IFM 41739 | Bark | China | + | - | - | F | |
IFM 41740 | Bark | China | + | - | - | F | |
IFM 41746 | Straw | China | + | - | - | F | |
IFM 41749 | Bark | China | + | - | - | F | |
IFM 41752 | Bark | China | + | - | - | F | |
IFM 41764 | Soil | China | + | - | - | F | |
IFM 41755 | Unknown | China | + | - | - | F | |
IFM 41765 | Soil | China | + | - | - | F | |
IFM 41778 | Straw | China | + | - | - | F | |
IFM 41872 | Soil | Colombia | - | - | - | N | |
IFM 41871 | Soil | Colombia | + | - | - | F | PV41 |
IFM 41879 | Soil | Colombia | + | - | + | FH | |
IFM 41881 | Soil | Colombia | + | - | - | F | |
IFM 41885 | Soil | Colombia | + | - | - | F | |
IFM 41884 | Cactus | Venezuela | - | - | - | N | |
IFM 41888 | Rotten wood | Venezuela | - | - | - | N | |
IFM 41887 | Rotten wood | Venezuela | + | - | + | FH | PV28 |
IFM 41886 | Cactus | Venezuela | + | - | + | FH | TH31 |
IFM 41892 | Soil | Venezuela | + | - | + | FH | TH35 |
IFM 41883 | Soil | Venezuela | + | - | - | F | |
IFM 41893 | Soil | Venezuela | + | - | - | F | |
IFM 41897 | Soil | Brazil | + | + | + | FGH | PV33 |
IFM 41898 | Soil | Brazil | + | + | + | FGH | PV34 |
IFM 41899 | Soil | Brazil | + | - | - | F | |
*CBS 273.37 | Human | Brazil | - | - | - | N | |
*CBS 400.67 | Soil | Brazil | - | - | - | N | |
*CBS 281.35 | Human | USA | - | - | - | N | |
*CBS 220.97 | Linden tree | USA | - | - | - | N | |
*CBS 840.69 | Decaying timber | Finland | - | - | - | N | |
*CBS 221.97 | Unknown | Uruguay | + | - | - | F | |
*CBS 223.97 | Human | USA | + | - | - | F |
*: P. americana, +: with insertion, -: no insertion, na: not analized.
List of ITS, 28S rDNA and intron sequences of P. verrucosa
Sample ID or entry name | Length (bp) | Splice positions | Accession number | |||||
ITS | 28S | Intron-F | Intron-G | Intron-H | positiona | positionb | ||
Yao | 535 | 3349 | AB550779 | |||||
a Position means relative to the 28S rRNA of P. verrucosa Yao strain and b position means relative to 23S rRNA of E. coli J01965.
Primers used for the amplification and sequencing of P. verrucosa
Primer | Sequence (5'-3') | 5' position* | Source | 5' position including ITS |
ITS1 | TCCGTAGGTGAACCTGCGG | -563 | White TJ, et al. [48] | 1 |
ITS3 | GCATCGATGAAGAACGCAGC | -309 | White TJ, et al. [48] | 255 |
NL1 | GCATATCAATAAGCGGAGGAAA | 39 | O'Donnell K [49] | 603 |
3PV26 | CCGTCTTGAAACACGGACC | 633 | This work | 1197 |
inFG-F | CCGAAAGATGGTGAACTATGCC | 795 | This work | 1359 |
inF-F | ACGTGCAAATCGATCGTCAA | 868 | This work | 1432 |
inF-R | CAAGGCCTCTAATCATTCGCT | 1009 | This work | 1573 |
8PV26 | GAACCTTTCCCCACTTCAG | 1487 | This work | 2051 |
11PV26 | AAGCCATAGGGAAGTTCCGT | 1525 | This work | 2089 |
9PV26 | GTCGTACTCATAACCGCAG | 1818 | This work | 2382 |
CA-INT-L | ATAAGGGAAGTCGGCAAAATAGATCCGTAA | 1881 | McCullough MJ, et al. [50] | 2445 |
2PV26 | TCCCGAAGTTACGGATCTA | 1918 | This work | 2482 |
16PV26 | CCCAACCCTTAGAGCCAATC | 1942 | This work | 2506 |
10PV26 | CCGTACCAGTTCTAAGTTG | 2089 | This work | 2653 |
inG-F | GATGGCCAGAAAGTGGTGTTG | 2130 | This work | 2694 |
inG-R | TAGGGACAGTGGGAATCTCGT | 2314 | This work | 2878 |
26S-INT3 | CTAGCGAAACCACAGCCAAG | 2323 | This work | 2887 |
CA-INT-R | CCTTGGCTGTGGTTTCGCTAGATAGTAGAT | 2343 | McCullough MJ, et al. [50] | 2907 |
inFG-R | GCTCTCCCACCTATTCTACACC | 2445 | This work | 3009 |
12PV26 | TGGTATTTCACCGGCGATTG | 2464 | This work | 3028 |
IGS1-M-R | CTGCCACAAGCCAGTTATC | 2805 | This work | 3369 |
IGS3-M | CTTCGATGTCGGCTCTTCCT | 2838 | This work | 3402 |
IGS-L | TAGTACGAGAGGAACCGT | 2991 | Williamson ECM et al. [51] | 3555 |
3IGS-PV | TCTAAGTCAGAATCCGTGCCG | 3090 | This work | 3654 |
5IGS1-PV | ACGAGCTACTGAGCGTAAG | 3318 | This work | 3882 |
6IGS-PV | GACCACAGTCAGGCTTACG | 3349 | This work | 3913 |
L2563 F | CACAGGGATAACTGGCTTGTGG | 2781 | This work | 3345 |
L2563R | ATCTGAATCAACGGTTCCTCTCG | 3018 | This work | 3582 |
* The 5' position is relative to the 28S rDNA sequence of the P. verrucosa Yao strain.
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Feb. 26, 2012
Site: Irvine, Calif. (Anteater Aquatics Complex)
Event: UC Irvine Invitational (Day 1)
Scores: #12 Michigan 9, #6 Hawai'i 8; #3 UCLA 13, #12 Michigan 3
Records: Michigan (5-6), Hawai'i (2-4), UCLA (10-1)
Next U-M Event: Sunday, Feb. 26 -- vs. Loyola Marymount - UC Irvine Invitational (Irvine, Calif.), 9:15 a.m. PST
IRVINE, Calif. -- The 12th-ranked University of Michigan water polo team opened day one of the UC Irvine Invitational with a 9-8 win over No. 6 Hawai'i before dropping game two on the day with a 13-3 loss to No. 3 UCLA on Saturday (Feb. 25) at the Anteater Aquatics Complex.
Kiki Golden (Danville, Calif./Monte Vista) scored four points on the day. Lauren Thatcher (Canyon Lake, Calif./Temescal Canyon) recorded her first career hat trick, with Lauren Colton (Mission Viejo, Calif./Tesoro) scoring one goal in each game.
In the first game of the day, Golden and Thatcher recorded hat tricks to guide Michigan to its second win all-time against the Rainbow Wahine. With the score tied at four during the intermission, U-M outscored Hawai'i 4-3 in the third quarter and used a late penalty to tough out the victory, with both teams scoring goals in the fourth quarter.
Offensively, Claire Ivry (Orinda, Calif./Miramonte), Audrey Pratt (Burlingame, Calif./Burlingame) and Colton each scored a goal, with Alex Adamson (Coronado, Calif./Coronado) posting nine saves in the cage.
UCLA's nine goals in the second half were the deciding factor in game two. After trailing 4-2 at the break, Michigan fell behind 8-2 at the end of the third period as a result of four unanswered goals in the period. U-M added one more goal in the fourth period but could not climb out the hole, with the Bruins adding five scores in the final frame.
Golden, Colton and Katie Hazelrigg (Rolling Hills, Calif./Chadwick) put Michigan on the scoreboard with a goal apiece. Goalie Morgan Turner (Mission Viejo, Calif./Trabuco Hills) returned to the pool, posting 11 saves.
Next, the Wolverines will start day two of the UC Irvine Invitational when they face No. 10 Loyola Marymount. The game is scheduled for 9:15 a.m. PST. Depending on the outcome, U-M will either face No. 9 San Diego State or No. 5 Arizona in its final game of the tournament.
By Period | 1 | 2 | 3 | 4 | F |
Hawai'i | 3 | 1 | 3 | 1 | 8 |
Michigan | 3 | 1 | 4 | 1 | 9 |
Hawai'i Goals: Amarens Genee 5, Emily Scott 2, Sivan Pardon
Saves: U-M 9 (Alex Adamson), HAW 5 (Serena Reid)
By Period | 1 | 2 | 3 | 4 | F |
Michigan | 1 | 1 | 0 | 1 | 3 |
UCLA Goals: Emily Greenwood 3, KK Clark 3, Gisselle Naranjo 2, Nicole Barker 2, Becca Dorst, Sarah Orozco, Emily Donohoe
Saves: U-M 11 (Morgan Turner 11), UCLA 9 (Dement 7, Wilkey 2)
Kiki Golden surpassed 100 career goals (102) after recording a hat trick against Hawai'i.
Michigan improves to 2-10 versus Hawai'i and falls to 0-10 against UCLA in the all-time series.
In the UC Irvine Invitational, Michigan now holds a 6-12 record.
Michigan now has defeated two ranked opponents this season and is now 2-6 against teams ranked in the top 20.
The win against Hawai'i was the first time Michigan defeated a team ranked higher in the national poll this season.
The last time Michigan defeated Hawai'i was on Feb. 7, 2010, in double overtime, 11-10.
The last time Michigan defeated a higher ranked team in the national poll was Jan. 22, 2011, when it handed No. 7 San Jose State a 9-7 loss during the Michigan Kick-Off.


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A shot taken of one-half of The Veronicas during their Baltimore show.
4 responses
Ashley Michele Ommen gave props (27 Jun 2008):
how has noone made a comment yet!!! Amazing shot!!
christine beaver said (28 Jun 2008):
i love this photo and i love jessie.
rita p said (28 Jun 2008):
omg i looove this shot :D amazing, jess = love
ana urrutia said (29 Jun 2008):
Lol!!is the best photo of jess form concert.


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View Bubba84's: | home | workshop | projects (5) | blog (0) | reviews (0) | forum topics (1) | buddies (1) | favorites (0) | activity log |
Remove An Uploaded Project - 2 replies I have two photographs of my work bench that are the same project. I uploaded (by accident) both photos as separate projects, not knowing that I should have uploaded both under the same project to show the progress as it went along. My question is, how do I remove the one project listed under my projects so it does not appear that I have two pro... |


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Left in the Dark: No One On Board
Average Rating 17 player reviews ( 4.8 out of 5.0 )
Help detective Charlotte Austin solve the mystery of the ghost ship and its missing crew!
Game Description
When private detective Charlotte Austin accepts her latest commission, she immediately knows it will be an unusual one. A bizarre letter from the Mayor of Port Providence arrives asking for help investigating the mysterious disappearance of a missing ship, which has suddenly reappeared without crew or cargo.
During her first attempts to solve the case, Charlotte realizes that the reports in the local press about an alleged curse may be more than local folklore. Her encounter with a hooded figure in the supposedly abandoned shipwreck, along with other seemingly impossible events, reinforce her belief that there is more going on in Portsmouth than meets the eye.
Detective Austin must discover the link between the abandoned ship, Devil Island, and the unsolved murder of a local family. Will she discover the curse that is dooming ships out of Portsmouth, and be able to stop it before more people die?
-Unique puzzles with varying difficulty levels
-Exciting storyline
-Stunning art
Most Recent Reviews To submit a rating or review, download this game
This is an HO Adventure game not a TM. It was good. Had a great story line and HO's are clear and easy. Mini games took a little time a patience but can be done. Enjoyed looking for objects to move on in this game. Hope they come out with a follow up .I want to know what happened to James to make him do such horriffick crimes and where is he now ???? Enjoy. See more See less
I really loved playing this game. I loved the music it fit really well with the feel of the game, the graphics were great as well and the story was good also, it almost scared me. The H.O. scenes weere fun too. The only complaint I have is I can't get enough. See more See less
Great Game. Graphics were great . It has casual play and Hints refill quickly. It has a map to help you. I love those. No running around. Story was good. Didn't have sound on. You can skip the mini games. Great HOG! See more See less
This was a fairly good game, Alot of back and forth though. But iWin has it categorized wrongly! It is not a time management game but a hidden object game. Some of the mini games are tough but you can skip after a decent period of time. Not a bad game even tho it's not my favorite genre. See more See less
This is not a TM game. It's an HO game. I played it just the same and enjoyed it. Lots of back and forth which I like. See more See less
Excellent fun. A really long game with twists and turns at every corner. I had to ask for help quite a bit, but that's just me. And the help was readily available. The characters were interesting as well and creepy at times. Thanks iWin, great game. See more See less


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The mod you are trying to view has ceased development and consequently been archived. If you are a member of this mod, can demonstrate that it is being actively developed and will be able to keep this profile up to date with the latest news, images, videos and downloads, please contact us with all details and we will consider its re-activation.
GQ is a Quake 1 Engine Refit. With plenty of fun changes, it'll make your eyes happy. Due to popular demand, the majority of new things are optional. Always in Development, drop me a line with suggestions.


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Affinity Group: South Asian Peoples
Affinity Bloc: South Asian Peoples
People Cluster: Tamil
Language: Tamil - (tam)
Alternate Name(s): Kayikolan, Kaikolar, Sengurithar, Sengundar Christian, Mudaliar, Segundar, Kerala Mudaliyar, Sengunthar, Karikalabhakthulu, Kaikali, Sengundam, Sangun
Primary Religion: Hinduism
Country: India
Also found in:
Population: 2,135,000
Global Population: 2,135,000
Progress of the Gospel: Engaged yet Unreached
Global Status of Evangelical Christianity: 2
Overview: The Kaikolar of India, numbering 2,135,000, are Engaged yet Unreached. They are part of the Tamil people cluster within the South Asian Peoples affinity bloc. This people group is only found in India. Their primary language is Tamil. The primary religion practiced by the Kaikolar is Hinduism, the predominant religious tradition of the Indian subcontinent. There are many forms of Hinduism, each with its own deities and beliefs.
Bible Translations: Available
Jesus Film: Available
Gospel Recordings: Available
Radio Broadcast: Available