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SDCC: "Batman: The Killing Joke" Cast & Crew Debuts Film at Comic-Con International
Can you handle a Previews preview on a weekday instead of a weekend? You're going to have to deal with it!!!!
Gail Simone is writing Tomb Raider (page 32). I'm not sure it's possible for me to have less interest in this, but I guess this is a thing, and I'm nothing if not comprehensive, so there it is! (26 February)
Dan Jolley and Jamal Igle are doing a new Terminator mini-series, Enemy of My Enemy (page 36). Once again, I'm not that terribly interested in this, but that's not a bad creative team. (19 February)
I have no idea what The Atomic Legion (page 38) is – Mike Richardson writes a story about old heroes who live in a secluded North Pole fortress and what they do when their benefactor is kidnapped – but it sounds wacky, and Bruce Zick is a good artist, so I might give it a chance. It's 30 bucks, though, so maybe Dark Horse will do a softcover version down the line? (30 April)
Tommy Lee Edwards, Noah Smith, and Dan McDaid are doing Vandroid, which is supposed to be about a movie from 1984 that was tragically lost and is now being "resurrected" in comic book form. The movie never existed, though, so I'm wondering why they're doing all the hoopla about it. I mean, it's clever, but not that clever. Still, that's not a bad creative team, although I'm not sure if Edwards is a decent writer or not when he's not drawing the book. (26 February)
Over on page 51, Brain Boy gets a trade. The story from Dark Horse Presents was pretty good, and Fred van Lente, Freddie Williams II, and R. B. Silva are a good team, so I'm might have to pick this one up. (16 April)
The Black Beetle: Necrologue #4 gets solicited on page 53. I don't mean to be snide, but the first issue is already at least 5 weeks late (I haven't checked to see if it's coming out this week), so shouldn't Dark Horse worry about getting the book going before they continue soliciting it? (26 February)
I'll probably get The White Suits (page 60) in trade, but it's probably going to be a good book. The few stories in DHP were pretty good, and the hook is intriguing – guys in white suits killing gangsters and the good guys looking for them – and more Toby Cypress art in our lives is always a good thing. (19 February)
There's yet another Lobster Johnson mini-series on page 61 – Get the Lobster. Tonci Zonjic is drawing it, so it will look good, but that just gives me an excuse to wonder where Where Is Jake Ellis? is. Remember that series? (5 February)
I've never heard of Someplace Strange by Ann Nocenti and John Bolton, but it's getting a new edition on page 70. According to the solicits, it's "a surrealist, new wave Alice in Wonderland!" Oh, okay then. I bet it looks great, and Nocenti from the 1980s is, from what I've heard, better than Nocenti of the 2010s, so if anyone has read this, sound off about it! (30 April)
Art Baltazar and Franco's Itty Bitty Hellboy gets a trade on page 73. Much like their Tiny Titans, this is amusing, but it's aimed squarely at kids. The trade is only 10 bucks, though, which is a pretty good price for five issues. (16 April)
The Metal Men show up in Justice League #28 (page 81), and it's pretty sad that the first thing I thought of was "At least Geoff Johns can't brutally kill them, or if he does, they can just get reassembled." Damn you, DC, for making that be my first thought! (19 February)
I'm really intrigued by Lois Lane #1 (page 103), even though it seems to be tied into Superman continuity. I don't know Bennett's writing (she's done some stuff for DC, but I haven't read it), but the solicit sounds interesting, and I give Emanuella Lupacchino's art a look even if I'm not terribly interested in the comic. It's a one-shot, so let's hope Bennett tells a complete story! (26 February)
That's such a weird-ass cover for Swamp Thing #28 (page 122):
It seems so … off. A sword-wielding fantasy-looking babe skewering our hero? Just … weird. (5 February)
In the Vertigo world, Rob Williams and Simon Coleby show up with The Royals: Masters of War (page 137), in which the royal family of England has superpowers but hasn't done anything to stop the Nazi blitz because of an old truce – but Prince Henry is having none of it! This could be intriguing, although I'm going to wait for the trade and see what the reviews say. We shall see! (12 February)
There's a new hardcover edition of Daytripper, the tremendous mini-series by Gabriel Bá and Fábio Moon, on page 142. I don't think it's ever been offered in hardcover before. It's 35 bucks, which is a bit pricey (the series was 10 issues, so it's not ridiculous, but still a bit spendy), but it is a wonderful series with gorgeous art, so there's that. (16 April)
DC continues to offer trades at pretty good value, as Death on page 142 contains both mini-series, plus a bunch of extras, giving us 320 pages for 20 bucks. They're good pages, too – both mini-series are pretty good, and the extra stuff is solid, too. If you don't have them, give this a look! (19 March)
Man, there's a Jack Kirby New Gods Artist's Edition on page 157. I'd have to sell one of my kids to a Somali warlord to afford that. Hmmm, I wonder …
The first issue of Mr. Peabody and Sherman came out last week, but IDW is already soliciting a trade! At 18 bucks, it's a bit expensive (and I think that's for only 4 issues, right?), but the first issue looked pretty keen. It's just another thing I have to ponder!
I haven't read Chuck Dixon's Airboy, but some of it gets collected in Airboy Archives volume 1 on page 183. Based on the artists, these are probably older stories, and it's 30 bucks for 300 pages, which for IDW is a pretty decent price. I'm intrigued, but not sure about this.
IDW has a Monkeybrain book on page 184: Mask of the Red Panda by Gregg Taylor and Dean Kotz. I like Kotz's artwork and I'm always a sucker for a good pulp adventure, but I don't think I know Taylor's work. Did anyone read this digitally and have an opinion on it?
Antony Johnston and Justin Greenwood's new series, The Fuse, gets offered on page 189. It's about a murder on an "orbiting energy platform," and if it's anything like Outland, I'm all over it. Even if it's not, Johnston is a good writer and Greenwood is not a great artist yet, but he's getting better, and his work looks better in color. So I have high hopes for this. (12 February)
Undertow (page 194) sounds interesting – an undersea dude who hates Atlantis explores the surface world looking for a way to live on land – and even though I don't know much about writer Steve Orlando, the art preview by Artyom Trakhanov looks incredible. I might have to get it just to stare at the pretty pictures. (19 February)
There's another Shaky Kane Elephantmen issue on page 218. Those are always a treat. Elephantmen has slipped far behind schedule, so I hope this comes out somewhat on time! (26 February)
As many people have noted, it's not the worst thing in the world for Marvel to start over with a #1 when they switch creative teams, but Wolverine #1 (page 2) takes it a bit far, as Paul Cornell is still writing the confounded thing. Ryan Stegman takes over on art, but given Marvel's shipping policy, he won't be able to draw it all, will he? This makes less sense than Marvel's usual renumbering policy, and that's saying something. (5 February)
Kelly Thompson's influence strikes again, as the descriptors for the Fantastic Four in the solicits for Fantastic Four #1 (page 4) are pretty funny, and thanks to the insightful Ms. Thompson, I noticed them: "brilliant" Mr. Fantastic (pretty standard), "ever-lovin'" Thing (also pretty standard), "hot-headed" Torch (also pretty standard), and … "compassionate" Invisible Woman. Bwah-ha-ha-ha!!! Oh, Marvel. One step forward, two steps back. (26 February)
If you're going to feature a Muslim superhero, like Ms. Marvel #1 (page 6) does, it's a good idea to get G. Willow Wilson to write it. That being said, I CANNOT BELIEVE MARVEL WOULD KOWTOW TO THE POLITICALLY CORRECT, ANTI-AMERICAN CROWD LIKE THIS AND PUBLISH THIS SHIT INSTEAD OF ANOTHER GOOD, WHOLESOME SUPERHERO COMIC STARRING A WHITE MALE!!!! MY CONGRESSMAN WILL BE HEARING ABOUT THIS, OF THAT YOU CAN BE SURE!!!! (5 February)
Rick Remender and Roland Boschi doing a 1960s espionage tale with Bucky Barnes sounds pretty dang cool, even though I'll probably wait for the trade on The Winter Soldier: The Bitter March (page 14). Although the way Marvel prices trades, maybe not. (12 February)
Al Ewing might be a good choice to write the adventures of Loki: Agent of Asgard, and Lee Garbett can be a very good artist. This is also (amazingly) 3 dollars, so I might have to check it out. I have heard that Loki is going to be bi-curious in this book, though. HOW DARE MARVEL PUBLISH A COMIC STARRING A DEGENERATE PERVERT INSTEAD OF ANOTHER SUPERHERO COMIC STARRING A WHITE MALE WHO LIKES CHICKS WITH BIG BOOBS!!!! MY CONGRESSMAN WILL BE HEARING ABOUT THIS, OF THAT YOU CAN BE SURE!!!! (5 February)
So on page 62, there's an OGN by Mark Waid, James Robinson, and Gabrielle Dell'Otto? It's Spider-Man: Family Business, and it's a bit expensive at 25 dollars for 112 pages, but Dell'Otto's painted work looks wonderful, and it's Waid and Robinson, after all. I love when Marvel does original graphic novels, because even more than DC's, they just seem so weird. What's the over/under for how long Dell'Otto has been working on this? Three years? (2 April)
There's the first trade of Superior Foes of Spider-Man on page 112 (and it's only 17 bucks for 6 issues, which is stunning for Marvel). I've heard lots of good things about this series, so I'm looking forward to this. (26 February)
Speaking of good price points, Daredevil: Dark Nights is 20 bucks for 8 issues. I don't know if this is any good (I know the Lee Weeks story looked amazing, but I don't know if the rest is decent), but I'm still probably getting this. (12 March)
Yes, we're past Marvel, which means it's time for the back of the book! Whoo-hoo!
On page 248, SLG offers Sanctuary volume 1 by Stephen Coughlin. I have reviewed this on this very blog, and it's pretty darned good. If that sways you at all, that is.
I'm not that interested in the 28 Days Later Omnibus from Boom! on page 282, but it's pretty neat that they're publishing it. It's 24 issues for 40 bucks, which isn't a bad deal.
Okko volume 4: The Cycle of Fire is offered on page 285. This is a pretty neat samurai/fantasy series. I should re-read the whole thing before this comes out, because I assume it's the last one.
Greg Pak and Mirko Colak fire up Turok: Dinosaur Hunter for Dynamite, because why not? The interior art looks pretty cool, and Pak is a decent writer, so I might have to check this out when it shows up. (5 February)
Gail Simone's Red Sonja gets a trade on page 300. This has looked pretty good, and the trade is 20 bucks for 6 issues, but I don't know if the story is any good. Is anyone reading it? (12 February)
I wonder if Dynamite is committed to collecting the entire late 1980s DC run of The Shadow, because they have The Shadow Master Series volume 1 on page 303. This is the first 6 issues by Andy Helfer and Bill Sienkiewicz, and it's pretty cool. I know the series got a bit weird as it went on, and I'd love it if it got collected so I wouldn't have to trawl through the back issues boxes. (26 February)
Fantagraphics offers Cannon by Wallace Wood on page 323. Shouldn't we call him "Wally"? It seems like everyone does, except Fantagraphics. Anyway, this sounds like a very cool strip from the 1960s, and I'm sure Greg Hatcher can tell us all how excellent it is.
Humanoids has Cape Horn, a story about characters in Tierra del Fuego at the end of the 1800s. This sounds like my kind of comic, although it's 40 dollars, so I'm not super sure about picking it up.
Joshua Fialkov and Joe Infurnari bring us The Bunker on page 332 from Oni. That's a good creative team, and the story – about a group of friends who find a strange bunker with hints about their future – sounds pretty keen. This was a digital comic, so I guess some people have already read it, but it's new to me! (5 February)
The trade of Atomic Robo and the Savage Sword of Dr. Dinosaur shows up on page 340 from Red 5 Comics. I'd take this with a grain of salt, as the schedule for the single issues has stalled a bit, but if you get Atomic Robo in trade, there it is!
Top Shelf offers The Bojeffries Saga on page 346. I've read a tiny bit of this, and I'm always interested in Alan Moore's work, so this is a no-brainer for me. Good for Top Shelf!
Well, that's about all we have this month. Alan Moore is a good place to finish, isn't it? I hope you have fun going through the solicits, and as always, let me know if there's anything I might have missed that you think deserves mentioning. We're all friends here!
Holy crap, Dynamite is reprinting the Helfer/Sienkiewicz/Baker Shadow series? I would like to give them all my money now, please. It's pretty easy to find those as back issues, but man oh man, that was my favorite Shadow series ever.
"This isn't the final cover, which is good because there ain't no string on that bow"
That's because he is so freakin cool he don't need no strings.
There's a Big Lebowski the Dude Talking Wacky Wobbler in the back that I've got to have. He does abide.
I will find a way to get the Jack Kirby New Gods Artist Edition. Like you, I may rob a bank.
I'm down for the Showcase Presents Jonah Hex, Volume 2.
I like a lot of the items you mentioned, especially the Shadow Master Series, which I have in single issues, but would love to have in a collected format.
So far, my shopping cart is way too big this month. It's gonna be tough to narrow it down.
Add another voice to the choir praising Superior Foes. It's been a genuine dandy of book so far, for me.
And I don't get the lols at describing Sue Storm as compassionate. Is that not a fair way to describe her?Hickman wrote her like that, but with a brave sorta compassion (when she tried to broker peace with the tribes of Atlantis comes to mind). But 'bravely compassionate' just sounds silly.
Definitely getting Kirby Artists Edition and Helfer/Sienkiewicz/Baker Shadow. And lots of other books you mention.
I haven't read Mask of the Red Panda, but Taylor also writes the Red Panda "radio" show, which has long been good. They just released the 100th episode at Decoder Ring Theatre, where you can listen to all of them for free.
In order to answer your query on Red Sonja and its possible quality: It's decent. The art is good at times, there is a strong direction in terms of characterization for Sonja herself, but the story isn't that amazing and there is a certain lack of interesting secondary characters overall. It's nice, but it certainly isn't the best thing Simone ever did in her career.
I decided to check out Vandroid to see what the story is behind the movie. Apparently all that survived was a script and some publicity materials…….The photos of which look to have been taken recently: http://vandroid.com/about/
Drawing a comic based on a "lost film" is an interesting concept, but unfortunately I think the backstory is just BS used to drum up interest in the project.
buttler: So far, it's just the first 6 issues. But yeah, that would be cool if they kept going.
Nicole: Ha!
DonW: I tend to stay away from the deep back of the book, so I missed that. Always nice to see Lebowski-related stuff, though.
dave: It's just such a "female" adjective. I very much doubt if you would ever hear a male character described that way. And, given that more than a few writers have described her as the most powerful member of the team, it would be nice if they would describe her that way. I know she is compassionate, so it's not necessarily the fact that they used it, just that it's such a "feminine" word.
Billy: Ah, very cool. Thanks!
Hugo: Good to know. I'll have to think about it.
Ian: Yeah, the backstory is made up. I don't know why they went that way – I assume the backstory will be part of the comic, but to extend it beyond that and pretend the movie actually existed seems excessive.
Anyone who mocks Susan Richards's compassion is also, by extension, dissing Lincoln's mother.
Calling him Wallace Wood shows respect. The only ones who ever called him Wally are the zillions of fans who never met him. His friends called him Woody. And that's how he usually signed his art.
buttler: Ha!
Jake: Interesting. I've seen him referred as "Wally" in a bunch of solicitations, but I gather the people writing those didn't do much research. I don't have a problem calling him "Wallace" – I was just wondering. That's cool to know.
Is it just me, or do those Metal Men look like somebody drawing a parody of what the Metal Men would look like in the new 52?
Also, Turok's bow does have a string. It's faint, but it's there. You can see it to the left of his ribcage and against his quiver to the right of his left arm.
I see a string on that bow. It's very very slight, but it's there. Check to the left of the left side of the quiver from our perspective and also through the right side of the quiver.
Chad: That's a pretty good description of the Metal Men!
Yeah, I do see the string. I think it's harder to see when it's printed in Previews, so I didn't even look too closely when I found that image on-line.
Chad: It's totally not just you. That really is how they look.
Ok, the cost-per-issue evaluation of trades makes no economic sense. $4/issue looks like a lot, but what if they are the best issues you have ever read. Cost-per-entertainment value-per-issue is a more sound measure.
Mudassir: I'm not commenting on the value. I think it's ridiculous that Marvel charges 4 dollars per issue, because none of their comics are worth that. What I think is ridiculous is that DC can somehow charge less money for reprints (which is what trades are) but Marvel usually doesn't. Yes, the value you get is important, but people often make decisions based on price. If Dynamite and Boom! can actually charge less for their trades (they don't always, but occasionally), then Marvel can. The reason I harp on price is because I think Marvel is full of shit when they charge $3.99 for a 20-page comic. DC has charged $3.99 for some of their books, but they usually had a back-up story. Now they're phasing those out, and the value you get for $3.99 has gone down. I don't care too much about price – if I really want to read something, I'll probably buy it – but I do think pointing out the price is not a bad thing. Everyone gets different entertainment value out of something, and occasionally I'll mention that the price might be high, but the product is really good. For things I haven't read, I can't make that claim. But I can point out that the way certain companies price their trades is weird, because it is.
The cost evaluation makes sense. For example, I don't typically buy a trade for more than $20. It would have to be an awesome book to convince me to spend more than that. I should know. I'm dealing poor. The only times I have spent more have been on some of those gigantic omnibus books that Marvel puts out. Even then, it was specifically for Tomb of Dracula and Howard the Duck. You bet your ass I wouldn't spend that much on anything less awesome.
For what it's worth, I think Someplace Strange is pretty great.
It looks interesting, of course, and when Bolton plays around with different art styles, it's really, really interesting, but Nocenti's story is just as good and weird and engaging. Mostly it's an adventure-story meditation on how eternally incompatible childhood is with adulthood and how there really isn't any commonality between the two — so, yeah, there's some Alice In Wonderland in it.
The most fascinating thing about the story is that as it ramps up to its (slight SPOILER) mostly happy ending, the only thing you feel is a crushing depression and, like, existential sadness. I'm not sure I've ever felt that way while reading a comic before; it's a disconcerting effect. I'd recommend it!
Also: although I think Nocenti's recent work is interesting but flawed (and her artistic partners have, at times, actively worked against her), her stuff from the 80s and early 90s is far, far better — unimaginably brilliant, in my opinion. (Why her Daredevil run, one of the greatest runs in comics, has such a spotty reprint history is beyond me.) But my point is this: Someplace Strange is THAT Nocenti.
Any extras in the hardcover of Daytrippers?
Oh, who am I kidding, I will buy this series yet again no matter what.
You know what? I'd love it if Dynamite could get Helfer and Baker back together again to finish up their SHADOW run that DC cancelled abruptly.
That is, if Dynamite continues to collect the remainder of that Shadow series, it would be really cool to see a reunion of Helfer and Baker.
Wouldn't it?
Thanks again to Greg for the promotion of Sanctuary Volume 1! If anyone is interested in reading it through Comixology, let me know and I'll spot you a few issues. Just 99 cents each and the first one is free! My email is firstname.lastname@example.com
"I know the series got a bit weird as it went on, and I'd love it if it got collected. "
And so my 1 man quest to get the final story completed…continues.
Is it just me, or do those Metal Men look like somebody drawing a parody of what the Metal Men would look like in the new 52?
It doesn't look like a parody of the New 52. It just looks like plain old New 52. The whole thing has been self-parody from the very start, to the point that it's almost impossible for a third party to parody.
I went ahead and preordered that Shadow collection and the Showcase Jonah Hex. Because I have no self-control.
Superheroes tend to be compassionate. Spider-Man is compassionate, Superman is compassionate. The Thing is compassionate!
I would have gone with "badass" for Sue myself. She's the badass one. That's her role within the FF, going back to the Kirby days when she beat up Doctor Doom with her bare hands that one time.
Marvel's numbering is all over the place. Nearly every issue is a new number 1, or a regular issue, but with a big number 1 on the page for some reason. Or its a point Now (.now) issue. WTF?!?
To top it off, they cancelled 2 books I read for 2.99 (X-Factor and Daredevil), only to reboot them at 3.99 a month or 2 later.
So I am now glad to say I don't buy any of their monthly anymore.
Yeah, I just was reading an interview with Howard Chaykin, who was a Wallace Wood assistant, and he says in there that it was either "Wallace" or "Woody", never "Wally".
Daredevil Dark Nights is well worth 20 bucks. The Lee Weeks story is one of the best of the year. The Lapham 2 parter was pretty decent stuff. I got the first issue of the Palmiotti written (drawn by…somebody whose name I forget), and while it was decent, it just wasn't as good as the other 2 stories. I'll be getting the last 2 issues of the series in the cheapo bin whenever they land there.
More babbling when I get my hands on a Previews. Didn't get to the store I usually get it from last week. But my my my, that Shadow trade….
The Fuse, gets offered on page 189. It's about a murder on an "orbiting energy platform," and if it's anything like Outland, I'm all over it.
Thought of OUTLAND but also of Rucka's WHITEOUT. I mean: homicide, hostile environment, workers and engineers in isolation…
What bugs me is the "half million people" part of the solicit (whether that's just going to be some big-city crime story, just located in space, sold as "BLADE RUNNER meets OUTLAND, baby!"), and the fact it seems an ongoing (instead of a mini or a series of minis), so I'll prolly just get a #1 sampler and see later if the first trade is worth it (Previews is a hostile environment too).
"compassionate" Invisible Woman. Bwah-ha-ha-ha!!!
Well, mebbe "compassionate" means "badass" nowadays, just like "pathetic" now means "appalling"? I mean, just think of Dubya's "compassionate conservatism" and what that turned out to be? Mebbe Invisible Woman is the George Bush to Mr. Fantastic's Dick Cheney, and compassionate is the new badass?
Besides, if "women should be seen and not heard" is demeaning, then "Invisible Woman", who is heard and not seen, surely is empowering? Marvel, such an early bastion of feminism!
But what if it's a congresswoman? (Not that there's anything wrong about having congress with a woman…)
SLG offers Sanctuary volume 1 by Stephen Coughlin. I have reviewed this on this very blog, and it's pretty darned good.
Sorry but even after checking your review I still felt forced to pass: supposedly it is my sort of murder mystery, the cute cartoony art and exagerated facial expressions would pull me out of it — as if DARK CRYSTAL was done in the style of THE MUPPET SHOW. Now, if only the art had been like the cover, which looks Scott Morse great…
However if you like that sort of setup, you may want to check out Adam Hines's great DUNCAN THE WONDER DOG which is kinda like a long-form ELMER as if drawn by Dave McKean and Chris Ware (or Douglas Paszkiewicz of Arsenic Lullabies). The first 400-page book was published in 2010 by Adhouse but it's also at http://www.geneva-street.com/duncanthewonderdog/ for free.
(The setup is real slow, there's a 50-page prologue of vignettes before the main stories begin! One could sample scenes like p. 67-75 about killing a sentient cow, p. 81-83 about a pet dog traumatized after learning how steaks are created, p. 130-138 about an ongoing terrorism investigation, p. 161-176 about the animal terrorists in hiding, or p. 215-223 about an ape working in politics talking at home with his human partner. They're some of the main threads, running concurrently and interspeded with vignettes.)
Anyway it's our world if animals were able to talk and fight for civil rights, some ala Martin Luther King, others ala Malcolm X. Its multi-threaded format is hostile to casual reading but it's as ambitious as overlooked, and even if the planned books 2 to 8 never come out it'll remain great.
This isn't the final cover, which is good because there ain't no string on that bow
You're complaining there's no string attached?
Fantagraphics offers Cannon by Wallace Wood on page 323. Shouldn't we call him "Wally"? It seems like everyone does, except Fantagraphics.
Well, people have a right to self-identify with Bill or William, Steve or Steven or Stephen, Greg or Gregory… Wood identified with Wallace, and signed "Wallace Wood" or "Woody" in his trademark blackletter. The "Wally" thing was imposed on him among those fandom things of the era used to manufacture the fairytales of "bullpens" of happy-campy creators. It's well-documented that Wood hated it in the same way Schulz hated the title "Peanuts" imposed on his strip: it lacked dignity and respect.
In 1975, Wood did a 3-page extreme self-parody of "My World" retitled "My Word", ending on a grotesque version of himself at his drawing table, saying to the reader, "My name is Spafon Gool" (NSFW, floating online at http://johnglenntaylor.blogspot.com.es/2010/04/nsfw-week-wally-woods-my-word.html among other places). But when it was translated in Europe in 1977, the rights-holder Flo Steinberg said Wood preferred for it to end with a translation of "My name is Wallace Wood".
let me know if there's anything I might have missed that you think deserves mentioning.
Oh, I dunno… Gilbert Hernandez apparently potboiling "zombies, mutants, drug lords, and gorgeous women"? (FATIMA: THE BLOOD SPINNERS, p. 59 at DH) JMS apparently trying to remake Kolchak as a girl? (APOCALYPSE AL, p. 202 at Image) Jonathan Hickman apparently hired just to kick-start a series and leave it at #7 to someone else? (GOD IS DEAD #7, p. 263 at Avatar) But seriously, folks…
– Some translations of stuff I've already read:
Cinebook p. 291 is doing the smart sci-fi series VALERIAN, now at VOLUME 6: AMBASSADOR OF THE SHADOWS GN which is among the best albums of the series (each is a separate story, like Tintin). All-ages but insulting to neither teens nor adults.
D&Q p. 317 is reoffering the complete two-volume AYA (LIFE IN YOP CITY and LOVE IN YOP CITY). It's semi-autobio slice-of-life with drama, comedy, and romance in Ivory Coast, Africa. As good as engrossing.
Yen Press p. 364 has the 3rd and last HC of THERMAE ROMAE (a 6-volume zany manga repackaged as three double-hardcovers), so it's now complete for sure in English. It's also a steep price at $40 each 400-page oversized hardcover (it's been a bestseller in Japan and Europe at half that price, though at manga size). I'd recommend borrowing the first hardcover from a library or someone to be sure you're not missing out.
– Various worthy resolicits, such as THE COMPLETE ZOT (p. 326, always a nice gift), and a revised edition of last year's THE LOXLEYS AND THE WAR OF 1812 (p. 341, a historically accurate retelling, with the fate of one Canadian family as the framing device; spoiler warning: it ends with the White House in flames).
– And then there's the strange case of PARIAH (p. 57 now at Dark Horse): a sci-fi story of children become geniuses after being cured genetically, who are eventually suspected of terrorist conspiracy and rounded up by the government in a satellite, with great art by Weldele. It's been looking interesting on the radar for years but its troubled publishing history is all over the map.
It's a 12-chapter graphic novel. Issues 1-4 sound a bit like Brian Wood's DEMO (cf. http://www.pariahonline.com/pariah-comic-issues/ and their summaries) but converging towards a unified narrative at #5 once they're all locked up in a satellite.
When it started in 2011, a positive review (cf. http://comicsworthreading.com/2011/07/31/pariah-1/ by Johanna) warned that the Dabel Brothers could hardly be trusted about release. Issues 1-4 were printed, but the Vol. 1 collection never materialized and was eventually cancelled.
Then in 2012 they tried to reboot it, replacing #5 with a Vol. 2 #1 (as seen at http://www.comicbookresources.com/?page=preview&id=12089 with good preview art), but they eventually cancelled the Diamond solicits. Apparently it's been completed in digital since.
And now in 2013, Dark Horse announced they'd publish the whole 1-12 as an OGN. But actually they're soliciting a so-called #1 (of 8) which really seems to be just vol. 2 #1 all over again (skipping the four DEMO-like intro issues and getting straight to the locked-in-satellite part). So DH's issues 1-8 would really be chapters 5-12 of the GN, maybe with some recap page? Not that they seem to be telling you that much, apparently.
Anyway, I'm still interested but not touching it in pamphlets: at this point they should solicit a damn hardcover and softcover of the complete story already, maybe right after a $1 sampler issue or something. But I think it's one to keep a hopeful eye on.
Simon: Phew! Thanks for the suggestions; I appreciate them. I've heard of Duncan the Wonder Dog but have never read it, so I'll have to check it out. I tend to skip Cinebook stuff, even though they're good, because I've gotten quite a few of them in the mail and I'm way behind on reading them, so I have to catch up. I've read one volume of Valerian, and I agree it's pretty good. I also tend to skip reprints (unless I don't know they're reprints), because I usually mentioned them the first time around. Zot, of course, is tremendous, and I enjoyed but didn't love The Loxleys and the War of 1812. Thanks for the information about Pariah – I saw it and was intrigued, as I like Weldele's work, but just forgot to mention it. Based on what you wrote, I may have to wait for the collected edition as well!
I'm also curious about The Mask of The Red Panda. Sounds pretty interesting.
I think you're being a little harsh on the Metal Men. The organic- metal look is very cool to me. From covers, it almost looks like the MM can change their respective appearances at will.
Comics Should Be Good accepts review copies. Anything sent to us will (for better or for worse) end up reviewed on the blog. See where to send the review copies.


text:
A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.
Author Summary
Mammals have two copies of each gene in their somatic cells, and most of these gene pairs are regulated and expressed simultaneously. A fraction of mammalian genes, however, is subject to imprinting—a chemical modification that marks a gene according to its parental origin, so that one parent's copy is expressed while the other parent's copy is silenced. How and why this process evolved is the subject of much speculation. Here we have shown that all the genes in one genomic region, Dlk1-Dio3, which are imprinted in placental mammals such as mouse and human, are not imprinted in marsupial (wallaby) or monotreme (platypus) mammals. This is in contrast to a small number of other imprinted genes that are imprinted in marsupials and other therian mammals and indicates that imprinting arose at each genomic domain at different stages of mammalian evolution. We have compared the sequence of the Dlk1-Dio3 region between seven vertebrate species and identified sequences that are differentially represented in mammals that imprint compared to those that do not. Our data indicate that once imprinted gene regulation is acquired in a domain, it becomes evolutionarily constrained to remain unchanged.
Citation: Edwards CA, Mungall AJ, Matthews L, Ryder E, Gray DJ, Pask AJ, et al. (2008) The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals. PLoS Biol 6(6): e135. doi:10.1371/journal.pbio.0060135
Academic Editor: Chris P. Ponting, Oxford University, United Kingdom
Received: February 8, 2008; Accepted: April 18, 2008; Published: June 3, 2008
Copyright: © 2008 Edwards 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: Research was supported by the EU Epigenome NoE FP6, the MRC, and the Wellcome Trust.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ECR, evolutionary conserved region; IG-DMR, intergenic differentially methylated region; LTR, long terminal repeat; miRNA, microRNA; snoRNA, small nucleolar RNA; UTR, untranslated region
Genomic imprinting is a process that causes genes to be expressed according to their parental origin and is evident in plants and mammals. Many imprinted genes are located in clusters regulated by a single imprinting control element, whose function across the whole imprinted domain depends on DNA methylation acquired differentially in the male and the female germlines [1]. It is not known how or why mammalian imprinting evolved; however, its emergence is associated with the evolution of a placenta [2,3], and the correct dosage of imprinted genes is important in prenatal growth, postnatal metabolism [4], and neurodevelopment [5]. Where tested, the majority of imprinted genes are expressed and imprinted, sometimes specifically, in the placenta [6], suggesting that even distantly related placental mammals such as metatherians (marsupials) will have imprinting, while oviparous mammals, the prototherians (monotremes), will not. Assessment of the imprinting status of a few individual mammalian imprinted genes is consistent with these data. The orthologues of four genes imprinted in mouse and human are clearly imprinted in marsupials [7–10], and no evidence of imprinting has been found in monotremes, although only three genes have been tested to date [8,11,12].
The Dlk1-Dio3 imprinted domain in eutherian mammals contains the protein-coding genes Delta-like homologue 1 (Dlk1), Retrotransposon-like gene 1 (Rtl1/Mart1), and the type 3 deiodinase (Dio3) expressed from the paternally inherited chromosome, and multiple long and short non–protein coding RNAs including microRNAs (miRNAs) and C/D small nucleolar RNA (snoRNA) genes expressed solely from the maternally inherited chromosome (Figure 1A). Seven imprinted miRNAs are located within anti-Rtl1, and over forty are located further downstream including within the miRNA-containing gene Mirg (Figure 1A). All of the genes in the domain are developmentally regulated and expressed in a range of embryonic and extraembryonic cells types with postnatal expression being found predominantly in the brain [13–15]. In mouse, imprinting is regulated by an intergenic differentially methylated region (IG-DMR), located 75 kb downstream of Dlk1, that becomes methylated during spermatogenesis but remains unmethylated in the maternal germline [16,17]. When a targeted deletion of the IG-DMR is inherited maternally, an epigenetic switch occurs causing the maternally inherited chromosome to behave like the paternally inherited chromosome; no effect is seen when the deletion is paternally inherited. The IG-DMR is also differentially methylated in human [17], and recently identified patients with deletions and epimutations in the DLK1-DIO3 region indicate that this element likely acts as the imprinting control element in human [18]. Tight linkage and strong conservation of Dlk1 and Dio3 is maintained in all vertebrates. The two genes are located 10.5 kb apart in Takifugu rubripes, approximately 370 kb apart in chicken, and 830 kb in human and mouse (Figure 1B).
(A) Schematic representation of the Dlk1-Dio3 domain in mouse showing genes expressed from the paternal chromosome (blue) and noncoding RNAs (red) expressed from the maternally inherited chromosome. The imprinting control region for the domain is the paternally methylated IG-DMR (circle). Also shown are differentially methylated regions in exon 5 of Dlk1 and the promoter region of Gtl2. Filled circles, methylated; open circlesn unmethylated. Not drawn to scale.
(B) The relative positions of DLK1 and DIO3 in vertebrates. The domain sizes were calculated from the start codon of DLK1 to the stop codon of DIO3. (Genome builds were human March 2006, mouse February 2006, opossum January 2006, chicken May 2006, and fugu October 2004).
To determine the sequence and organization of the region in marsupial and monotreme mammals, we cloned and sequenced the region between DLK1 and DIO3 in the platypus, Ornithorhynchus anatinus, and the tammar wallaby, Macropus eugenii. Bacterial artificial chromosome (BAC) clones containing the orthologous DLK1 and DIO3 genes were identified [19]. Thirteen overlapping wallaby BACs and seven overlapping platypus BACs were isolated from genomic libraries, then initially characterized using a parallel landmark content mapping and fingerprinting strategy [20], and sequenced (Figure S1 and Table S1). This genomic sequence represents complete coverage of the domain in both species and was generated independently of the whole-genome sequencing projects for these organisms. The wallaby sequence is 1,510.8 kb and slightly smaller than that of the South American marsupial Monodelphis domestica (1,637.8 kb plus 26 gaps). The marsupial region is therefore approximately twice as long as its eutherian orthologue (Figure 1B). The region in platypus is 594.8 kb, which is 28% smaller than in mouse.
For both wallaby and platypus, DLK1 and DIO3 genes were identified, cDNAs characterized, and the genes subjected to imprinting analysis (Figure 2 and Figures S2 and S3). For wallaby, fetal tissues, yolk sac placenta, and pouch young samples were dissected. Platypus fetal material is unavailable, so the analysis was conducted on primary adult skin fibroblasts cultured from two male and one female platypus; therefore the analysis in that species is limited. Several single nucleotide polymorphisms (SNP) (Figure 2A) were identified for DLK1 from wallaby tissues that included one sample (2386) from a homozygous mother allowing allele-specific activity to be determined. Both maternally and paternally inherited alleles of DLK1 were expressed in all wallaby fetal, extraembryonic, and pouch young tissues analysed (Figure 2A and Figure S2C). Similar SNP and restriction fragment length polymorphism analysis showed biallelic expression of DLK1 in platypus (Figure 2B).
(A) DLK1 is biallelically expressed in tammar wallaby. The imprinting status of wallaby DLK1 was determined by analyzing cDNAs shown here from three individuals (638, 788, and 2386) heterozygous for a G/A single nucleotide polymorphism (SNP) in exon 4 at 374 bp from translational start. Biallelic expression was observed in yolk sac placenta (YSM), fetal head, fetal tail, and pouch young (PY) body. Results were confirmed with three further SNPs in the 5′ UTR (Figure S2).
(B) DLK1 is biallelically expressed in platypus. An A/C SNP was identified in the 3′ UTR of the platypus DLK1 gene 1,323 bp from the translational start. Sequence analysis of cDNA generated from an informative platypus primary fibroblast cell line demonstrated biallelic expression. The C allele of the SNP introduces an NlaIII into the region. RFLP analysis confirms biallelic expression of platypus DLK1.
(C) DIO3 is biallelically expressed in the platypus. Two polymorphisms in platypus DIO3 were identified in two different primary fibroblast cell lines—a G/C SNP and a 64 bp indel. RT-PCR analysis demonstrates biallelic expression.
(D) Two polymorphisms were identified in wallaby DIO3, a CTT indel and a G/A SNP. Preferential expression was observed from the –CTT/G allele, which was particularly evident in yolk sac placenta samples.
(E) Quantitative RT-PCR was used to assess the expression from each DIO3 allele in 12 different heterozygous individuals compared with a standard curve of two gDNA mixed at different ratios. Genomic DNA from all individuals was also tested and compared to the standard curve. Where more than one cDNA was analysed the data were combined and ± standard error are shown. All tissues tested displayed biased expression of the –CTT allele regardless of its parent of origin. BYS, bilaminar yolk sac; TYS, trilaminar yolk sac; YS, yolk sac; and mat, maternal gDNA. The maternal genotype for each individual is are shown in parentheses.
Two polymorphisms were identified in wallaby DIO3, a G/A SNP at nucleotide 94 in the coding region of the gene and a CTT insertion/deletion (indel) at nucleotide 1,187 in the 3′ untranslated region (UTR) (Figure 2A and Figure S3). Both polymorphisms were present in nine animals, suggesting co-segregation of the variant alleles. Direct sequencing of cDNAs amplified across both polymorphisms indicated there was preferential expression from the G/-CTT allele (Figure 2D). Quantitative real-time, reverse-transcriptase PCR (RT-PCR) proved that wallaby DIO3 was expressed from both parental chromosomes. However, an allelic bias towards the -CTT allele was observed in all samples tested regardless of parental origin (Figure 2E). Expression analysis of two polymorphisms in platypus DIO3 confirmed biallelic expression in this species (Figure 2C).
Comparative sequence analysis of the Dlk1-Dio3 genomic landscape between eutherian and other noneutherian mammals can identify the dynamic changes that are associated with and have the potential to contribute to imprinting. Figure 3A and Table 1 show the relative GC and repeat sequence content of the region in seven genomes; three eutherian species (human, mouse, dog), two marsupials (opossum and tammar wallaby), one monotreme (platypus), and one bird (chicken). The eutherian GC content, %CpG and number of CpG islands was significantly higher than the genome average (p < 0.01 using Chi-squared test) in contrast to marsupial and monotreme mammals, and chicken, that all lack imprinting at this domain. Repeat content was analysed using the most recent previously unreleased platypus repeat database (kindly provided by R Hubley, Repeatmasker). Eutherian LINE content is consistent with the genome-wide average; however, there is a paucity of LINEs in the region between Dlk1 and Mirg (miRNA-containing gene) in the eutherians (Table 1). The majority of repeats identified in the DLK-DIO3 domain in the marsupials are LINE1 repeats. This is consistent with the high number of LINEs identified in the opossum genome and suggests that expansion in the DLK1-DIO3 region, as in the marsupial genome as a whole, is due to LINE1 insertion. The opossum region has a slightly larger proportion of SINEs than expected from the genome average. The SINE content is also greater in the tammar wallaby, although the whole-genome sequence for this species is not currently available for comparison. The relative repeat content in platypus is greater than eutherians despite the region being smaller in this species (Figure 3A). The majority of repeats in the platypus DLK1-DIO3 region are SINEs and the more ancient LINE2s. Interestingly; there is a notable absence of long terminal repeat (LTR) elements at this locus in platypus (Figure 3A). The chicken region is devoid of any SINE elements which is consistent with the whole genome analysis of this species. Hence platypus and marsupials have greater SINE content in the domain than do the eutherian mammals with imprinting. This is consistent with the SINE depletion previously reported when comparing imprinted with nonimprinted domains in mouse and human [21,22]. Together, these findings indicate that selection against SINE repeats is an evolutionary feature of imprinted domains (see Discussion).
(A) Repeat content of the DLK1-DIO3 region in seven vertebrates. The region in both marsupials contains greater than 60% repeats, most of which are LINE1s. The platypus region contains approximately 50% repeats—this is a higher proportion than identified in the eutherian domain despite the region being 28% smaller in platypus. The platypus domain is depleted in LTR repeats.
(B) Distribution of the 141 ECRs identified. ECR groups are arranged according to the sub-classes of vertebrates they are identified in. Vertebrate: identified in at least one eutherian, one marsupial, platypus, and chicken. Mammalian: identified in at least one eutherian, one marsupial, and platypus. Therian: identified in three therians including one eutherian and one marsupial. H, human; M, mouse; D, dog; W, wallaby; O, opossum; P, platypus; C, chicken.
GC and Repeat Content of the Dlk1-Dio3 Region in Vertebrates
Detailed comparative sequence analysis was conducted between the Dlk1-Dio3 domain in the seven vertebrates. Using a threshold of 55% nucleotide sequence identity over 80 bp, which recognizes the Dlk1 exons in all seven sequences, 141 evolutionary conserved regions (ECRs) were identified across sub-groups representing eutherians, marsupials, platypus, and chicken (Table S2). Of the 141 ECRs found, 22.7% (31) were common to all seven vertebrates, 15.6% (22) were common to all mammals, and another 16 were found in all therian mammals. Six were found only in platypus and chicken. Figure 3B illustrates the number of ECRs arranged according to the sub-classes of vertebrates in which they are identified. In mammals, 27.7% were identified in at least one eutherian, one marsupial and platypus, whereas 24.8% were found in at least one species representing each therian infraclass. Although the greatest number of ECRs is found within the mammalian species, the more ancestral ECRs (the 31 found in all species studied) are on average larger, having a mean length of 494 bp compared with the mean length of all ECRs at 340 bp and suggesting greater functional constraint.
We used the 31 ECRs found in all vertebrates to align the Dlk1-Dio3 domain and subdivide it into 30 inter-ECR zones for further comparative analysis (Figure 4A). Exons of Dlk1 and Dio3 are represented by vertebrate ECRs 1–3 and 30–31, respectively. The intergenic distribution of the ECRs is not uniform throughout the domain with two-thirds being located in the 3′ half of the domain. One of the ECRs, approximately 3 kb upstream of DIO3, contains a highly conserved putative CTCF binding site in all therian species.
(A) ECRs identified in all seven species are shown as blue (Dlk1 and Dio3 exons) or black lines. The ECRs are linked to their orthologues in the neighbouring species in order to illustrate the repeat content and relative expansions/contraction within each sequence.
(B) The length of each inter-ECR zones from vECR1 (DLK1 exon 3) to vECR31 (DIO3) as a proportion of the length of the domain in eutherians, marsupials, platypus, and chicken. Zone 1 = vECR1–vECR2, zone 2 = vECR2–vECR3, etc. Mean ± standard error for the three eutherians and the two marsupials are shown.
The amount of sequence in each of the 30 inter-ECR zones relative to the overall size of the domain was quantified for each vertebrate (Figure 4B). This provides a measure of the overall expansions/contractions between species. The regional changes between marsupial, monotreme, and eutherian mammals across the domain are not uniform. The most striking differences between the mammals lie in zone 3 (between vECR3 and vECR4), zone 6, and zone 7. Zone 3 which is located between the last exon of Dlk1 and the conserved intron 5 region of Gtl2, is expanded in eutherians (Figure 4B). This expansion does not appear to be caused by LINEs, because LINE1 and LINE2 repeats are equivalently represented in eutherians and marsupials (Figure S4). As shown, this zone contains a higher proportion of SINE elements than reported for the whole genome and compared with the entire domain. However, the increased SINE content does not explain the expansion of zone 3, which is due to the acquisition of unique sequence, including the imprinting control region (the IG-DMR) and presumably other eutherian specific regulators. In contrast, eutherian zone 6, located between Gtl2 and Rtl1, is smaller than in marsupials, platypus and chicken, implying either that it contracted or that it is resistant to expansion. This latter explanation is favoured, because in marsupials expansion is predominantly due to LINE1s, and in platypus to LINE2 repeats and SINEs. This exclusion suggests an important previously unrecognized eutherian specific function for that zone (Figure 4).
The eutherian specific expansion of zone 7, as for zone 3, is not associated with the insertion of repetitive sequences, compared with marsupials. Rather, zone 7 represents the region located between Rtl1 and Mirg, which, in eutherians, contains approximately 50 miRNA genes and three clusters of C/D snoRNA genes, all expressed from the maternally inherited chromosome [23]. With one exception (see below), our analysis failed to find homologous sequences in marsupials, platypus, or chicken. Instead, the zone contains LINE1 repeats in marsupials and as before, LINE2s and SINEs in platypus (Figure S4). Therefore eutherians acquired transcribed non–protein coding RNAs in a zone that appears resistant to expansion by LINEs and SINEs. Interestingly, the acquisition of snoRNA genes in the imprinted Prader Willi-Angelman syndrome locus also corresponds to the acquisition of imprinting [12].
In the mouse, all the imprinted non–protein coding transcripts in the domain require the imprinting control element and sequences 5′ to Gtl2 for their activity on the maternally inherited chromosome. They are all expressed in the same orientation, and data suggest that they are at least in part associated with a single long transcription unit [17,24]. ECRs specifically associated with Gtl2 were identified by phylogenetic footprinting (Figure 5A). Two approaches were undertaken to determine whether GTL2 and other non–protein coding transcripts were present within the domain; expression analysis of DLK1-DIO3 intergenic ECRs and the amplification from cDNA of randomly selected sequences from the wallaby region (Figure S5A and Table S3). Five mammalian ECRs were found in the vicinity of Gtl2, of which three were common to all vertebrates; one corresponds to exon 5 of NM_144513 (ECR19), and the remainder appear to be intronic. One of the intronic ECRs (ECR18) was previously identified in intron 8 of Y13832 [22]. An additional ECR (ECR14) located close to exon 1 was identified and found to be inverted in eutherians (Figure 5A). This and the three vertebrate ECRs were expressed at very low levels in wallaby tissues, with no transcriptional activity from the other two. RT-PCR analysis of 29 additional, randomly selected sequences in wallaby located between Gtl2 and Mirg identified weak transcriptional activity from five sequences, including one mammalian Mirg-specific ECR (Figure S5). Quantitative RT-PCR comparing the relative expression of ECR19 and one of the random sequences (Ran3) with DIO3 expression in the same samples confirms expression from the GTL2-like locus in marsupials is between 1.1 × 10−4 and 4.2 × 10−4 lower in fetal head and pouch young brain (Figure 5). Polymorphisms located in ECR19 and the MIRG-ECR were used to demonsrate that this low level of transcription is biallelic (Figure 5B and Figure S4).
(A) Identification of ECRs in the Gtl2 region in noneutherians. mLAGAN and zPicture alignments of mouse Gtl2 with human, dog, wallaby, opossum, platypus, and chicken are shown. Four intronic ECRs are identified and one (ECR19) aligns within exon 5 of NM_144513. ECR14 is inverted in the eutherians and was only identified using the zPicture alignment. Weak expression was identified for ECRs14, 15, 18 and 19. RT-PCR for ECR19 in fetal head and pouch young body is shown.
(B) Weak expression from ECR19 in tammar wallaby fetal head and pouch young. An A/G SNP was identified in ECR19 and biallelic expression was observed.
(C) The expression ratio of ECR19 and Random Primer set 3 relative to DIO3 in fetal head and pouch young brain as calculated by quantative RT-PCR.
(D) A region orthologous to the retrotransposon-derived gene Rtl1 was identified in marsupials. The mLAGAN algorithm was used to align human RTL1 with mouse, dog, wallaby, and opossum. Regions with homology of >55% over 80 bp are shown in blue. Regions of human RTL1 with homology to the Sushi-ichi domains are highlighted. Homology between the eutherian and marsupial regions indicates that RTL1 inserted into the region before the divergence of eutherians and metatherians.
It was of particular interest to determine whether the protein-coding, retrotransposon-like gene Rtl1 (also known as Peg11/Mart1) was present in non-eutherian mammals. Rtl1 is a member of the Ty3-Gypsy family of LTR retrotransposons with closest similarity to the Sushi-ichi class [25]. In mouse and human, it has lost its LTRs, encodes a protein essential for normal placental development and fetal growth and viability (M. Ito, A. Ferguson-Smith, unpublished data, and [26]), and is expressed from the paternally inherited chromosome. Its levels are regulated by miRNAs processed from an antisense transcript on the maternally inherited chromosome that are 100% complementary to the Rtl1 mRNA (Figure 1A) [17,27,28]. Another member of this family, Peg10 located on mouse Chromosome 6, was recently shown to be imprinted in wallaby fetus and placenta (but is absent in the platypus), and its repression on the maternally inherited chromosome is associated with differential methylation in the body of the gene [9]. We could not demonstrate RTL1 sequences in the platypus or chicken domain. However, we did find sequences related to Rtl1 in the appropriate position in marsupials but, interestingly, it is extensively degraded with very few regions of homology remaining (Figure 5D). No expression of the most highly conserved region was found in fetal and pouch young tissues (Figure 6A). This suggests that Rtl1 retrotransposed into the locus prior to the divergence of marsupial and eutherian mammals and, in the absence of functional selection, it degraded in marsupials but acquired a growth regulatory function in eutherians coincident with the evolution of imprinting.
(A) Hypermethylation was observed in both the wallaby and platypus DLK1 exon 5 regions. Wallaby genomic DNA from d23 RPY fetus (gDNA) and wallaby sperm gDNA was digested with XbaI (Xb), further digested with HpyCH4IV (Hy) and analysed by Southern blot hybridisation using MeDLK1Ex5 as a probe. Platypus gDNA was digested with StuI (St) and further with MspI (Ms), HpaII (Hp), and HhaI (Hh) and analysed by Southern blot hybridisation using OaDLK1Ex5 as a probe.
(B) A map depicting the HpaII and HhaI sites and methylation status in Dlk1 exon 5. Black circles indicate methylated sites, white circles unmethylated sites, and half black circles indicate partial methylation. CpG islands in the region are shown as grey boxes.
(C) The Dio3 promoter region is unmethylated in both wallaby and platypus. Wallaby fetal head gDNA was digested with HindIII and further with MspI (Ms), HpaII (Hp), and HhaI (Hh) and hydridised with MeDIO3CpG. The methylation-sensitive HpaII and HhaI tracks exhibited full digestion indicating the region is unmethylated. Platypus gDNA was digested with XbaI (Xb) then with MspI (Ms), HpaII (Hp), HhaI (Hh), or SmaI (Sm) and hydridised with OaDIO3CpG. High CG content results in many HpaII and HhaI fragments, which are unmethylated and too small to be resolved on this filter. The smallest SmaI site expected was identified, showing the platypus Dio3 promoter is unmethylated. Control hybridisation with a OaDIO3 promoter proximal probe identified a fully methylated XbaI fragment of >3 kb in the HpaII, HhaI, and SmaI tracks, confirming the integrity of the genomic DNA in these tracks (unpublished data).
A number of miRNAs that are antisense to Rtl1 are transcribed from the maternal chromosome in eutherians. Using the miRNA prediction programme miR-abela [29], no miRNAs were found to be conserved between all vertebrates, and none were conserved between eutherians and marsupials. A single predicted miRNA was conserved between the marsupials (74% identity) (Figure S6B and S6C). Interestingly, this was located in the vicinity of the eutherian miR127, which is transcribed antisense to Rtl1 and along with seven others, contributes to the stability of the Rtl1 mRNA through an RNAi-dependent mechanism [27]; a function that would not be evident in marsupials that lack this gene. The sequence of the predicted processed miRNA from marsupial miR127 though common to both marsupials is less similar to eutherians and RT-PCR analysis failed to amplify the primary transcript or predicted hairpin from wallaby fetal head or pouch young brain cDNAs (Figure S6D). These data suggest that this is not a functional miRNA, and sequence similarity is due to miR127 being located within RTL1.
A small number of conserved CpG islands and CpG-rich regions were found to be shared between eutherians, marsupials, and platypus and their methylation status was determined. They included the promoters of Dlk1 and Dio3 and the differentially methylated region in the last exon of eutherian Dlk1, known as the Dlk-DMR [16,30]. Each region was analysed by methylation-sensitive Southern blots with genomic DNA from platypus and wallaby and from wallaby sperm. Results are shown in Figure 6. The ECR at intron 5 in Gtl2 (Y13832) is CpG-rich, and this too was analysed. As in eutherians, the DLK1 and DIO3 CpG-island promoters are completely unmethylated on both parental chromosomes. The Gtl2 ECR is partially methylated on both parental chromosomes in mouse, and has the same pattern in platypus and wallaby. In mouse, the Dlk-DMR is hypermethylated on the paternally inherited chromosome and in sperm, and hypomethylated on the maternally inherited chromosome [16,30]. Platypus and wallaby genomic DNA showed hypermethylation of the locus similar to that seen on the paternal chromosome in the mouse. Wallaby sperm was also hypermethylated. This suggests that the methylation state of the mouse paternal chromosome resembles the methylation state of the mammalian domain prior to the emergence of imprinting and implies that hypomethylation of the maternal chromosome evolved with imprinting.
In eutherians, Dlk1 and Dio3 are developmentally important genes that are expressed in numerous embryonic and extraembryonic tissues. Here we have shown that DLK1 and DIO3 are both biallelically expressed in marsupial fetus, placenta, and neonatal pouch young. DLK1 was recently shown to be expressed biallelically in adult brain, liver, and kidney in the South American marsupial, Monodelphis domestica; however, analysis of imprinting in embryonic and extraembryonic tissues was not conducted in that study [31]. We also demonstrate biallelic expression of both genes in platypus. Because fetal material is not available, biallelic expression of these genes during platypus development can only be inferred. Together, our results indicate that imprinting of the whole DLK1-DIO3 domain evolved after the divergence of metatherian and eutherian mammals.
Comparative sequence analysis of the DLK1-DIO3 region in seven different amniote vertebrates (representing Eutheria, Metatheria, Prototheria, and Aves) demonstrates that the overall genomic landscape in this region is GC-rich in eutherians but not in the other species studied. It has previously been postulated that GC-rich isochores in eutherians were once located on GC-rich microchromosomes in the ancestral amniote [32]. The elevated GC content in eutherians but not in the noneutherian species suggests that the increase occurred in eutherians rather than existing as an ancient isochore.
A number of results suggest that the DLK1-DIO3 is a recombination hot spot and under purifying selection in eutherian species where it is imprinted. First, elevated GC content correlates with increased levels of recombination [32]. Second, the introns of DLK1 are shorter in the eutherians than in the noneutherian species (Figure S2B), and decreased intron length is associated with high recombination rates [33]. Third, the reduced SINE content in the eutherian indicates the region is under purifying selection, especially because SINEs are usually associated with GC-rich regions. Interestingly, the region between vertebrate ECR1 and ECR8, which encompasses Dlk1, Gtl2, Rtl1, snoRNAs, and miRNAs, is particularly devoid of LINEs, indicating that this region is under even greater constraint (Table 1 and Figure 4A). Finally, the eutherian DLK1-DIO3 regions are also all located close to the telomeres, whereas in noneutherian species, they are located mid-chromosome [19]. A correlation of elevated recombination levels at sub-telomeric regions has previously been reported [34–36]; however, it is possible that this sub-telomeric position is the result of increased breakage in GC-rich regions [37]. Imprinted domains have previously been shown to be associated with elevated GC content [38–41], short introns [42], and reduced SINE content when compared to nonimprinted regions in eutherians [21,43]. Our finding that this comparison can be extended to the same domain between mammals that imprint and those that do not strongly suggests that imprinted domains are under purifying selection perhaps to constrain domain size such that cis-acting elements can function correctly.
None of the ECRs maps to the position of the eutherian imprinting control element. Whether any of the ECRs plays a functional role in the regulation of the domain is currently under investigation. Those specific to subgroups such as oviparous vertebrates, or the sixteen ECRs specific to therian mammals, might relate to the regulation of specific functions such as the development of extraembryonic structures in therians.
Expression analysis has provided evidence that Gtl2 and other noncoding transcripts existed throughout amniote evolution, suggesting that Gtl2 did not arise from an eutherian-specific retrotransposition event that triggered imprinting at the domain as has been previously suggested [31]. Our results show that weak regional non–protein coding transcriptional activity can occur in some places across the domain in noneutherian mammals and suggest that the process repressing the protein-coding genes on the maternal chromosome in eutherians (driven by the imprinting control region upstream from Gtl2) facilitated stronger expression from these non–protein coding transcripts. The appearance of functional miRNAs and C/D snoRNAs within the locus may therefore have been a consequence of the acquisition of imprinting with the strongly expressed Gtl2 gene, providing an ideal host transcript. It is not known whether the duplications that gave rise to the miRNA clusters occurred before or after evolution of imprinting at the locus. Interestingly, a role for these miRNAs in the trans-regulation of neural and placental processes has been inferred [44]. A functional role for these transcripts in the regulation of the neighbouring imprinted protein-coding genes also cannot be ruled out. Furthermore, the emergence of a regulatory relationship between RTL1 and its reciprocally imprinted miRNA-containing antisense transcript is also intriguing. In contrast to the more distal miRNA clusters to which they are not related, these seven anti-RTL1 miRNAs are not likely to have arisen through duplication/divergence events. Rather, these may have evolved as a host defence mechanism associated with the retrotransposon properties of RTL1, and evolved with it to modulate its expression [27] as it acquired an endogenous function.
During the course of evolution, the genomic landscape of the Dlk1-Dio3 region has undergone a number of changes (Figure 7). Most significantly, the region has become imprinted. This analysis has proven that imprinting in this domain emerged after the divergence of marsupials and eutherian mammals. This provides evidence that mammalian imprinting evolved at different loci at different times in response to selective pressures acting on different domains, suggesting an adaptive process. Prior to the divergence of metatherians from eutherians, the Sushi-ichi retrotransposon Rtl1, inserted between DLK1 and DIO3, gained no function and was degraded in marsupials. In marsupials, the region expanded 2-fold through the insertion of LINE repeats. As the eutherian lineage evolved through selective regional changes, Rtl1 evolved into a new gene acquiring a vital function in growth and development. This gain of function may indeed have driven imprinting at the domain, conferred through the acquisition of the imprinting control element. Gtl2 and associated transcripts became up-regulated on the maternal chromosome in eutherians, and miRNAs and C/D snoRNAs specifically evolved in the region. Once imprinted, gene expression was fixed in the region it underwent purifying selection, correlating with an increase in GC content, reduction in Dlk1 intron size, and selection against SINE and LINE insertions. Comparison of these results with similar detailed analyses on domains acquiring imprinting prior to the divergence of marsupials and eutherians will provide further insight into the relationships between dynamic changes in genomic landscape and the evolution of imprinting.
Schematic illustration of the evolution of the Dlk1-Dio3 domain in mammals. RTL1 retrotransposed into the region before the divergence of the eutherians and metatherians. In the marsupial lineage, RTL1 did not gain a function (or lose it) and became degraded. The region expanded approximately 2-fold in the marsupials; this expansion is mainly due to the accumulation of LINE1 repeats. The snoRNA and miRNA clusters arose after eutherian diverged from marsupials but before the mammalian radiation which took place around 98 million years ago. The eutherian region has also evolved many genomic features associated with imprinted clusters. The entire domain has become increasingly GC-rich, whereas a decline in GC content is the general trend in eutherian genomes. There are fewer SINEs than expected in the region, and the introns of the DLK1 transcript have become shorter. Finally the region has a sub-telomeric position within the eutherian genome whereas in monotremes and marsupials it is in the middle of the chromosome arm. Not drawn to scale
Materials and Methods
Expression analysis.
RNA was extracted using the GenElute mammalian total RNA miniprep kit (Sigma) following the manufacturer's protocol. cDNA was synthesized using Superscript III RNase H− Reverse Transcriptase (Invitrogen) following the manufacturer's instructions. The RT-PCRs were primed using random hexamer primers or the following gene-specific primers; platypus DLK1 5′-GAACGTTTATTTTACAAAAGATAGCTG-3′, wallaby DIO3 5′-CGGGCACTCACAGAGTTACA-3′, and platypus DIO3 5′-GACTCCGTCTCCGAGAACAT-3′, and 5′-TGAACATCTTACAAAAACCAACAAA-3′. cDNA was amplified using Hot Start KOD polymerase (Novagen), PCR conditions are as described in [19]. For particularly GC-rich regions (e.g., platypus DIO3) 1× Polymate (Bioline) was also added to the PCR reaction. Primer sequences and annealing temperatures can be found in Table S3). PCR fragments were gel purified using Qiaquick Gel Extraction Kit (Qiagen) and sequencing was performed.
For ECR and random sequence expression analysis, cDNA was generated as above using random hexamers and PCR amplification performed using either Hot Start KOD polymerase (Novagen) or Taq polymerase (Bioline), using conditions described in [19]. The primer sequences and annealing temperatures can be found in Table S4.
Allelic discrimination quantitative RT- PCR.
Custom TaqMan assays were produced using the Assays-by-Design facility at Applied Biosystems. 1 μl of cDNA was amplified in a 12.5-μl reaction 1× TaqMan Universal PCR Master Mix (Applied Biosystems) and 1× specific assay as per the manufacturer's instructions. CT (threshold cycle) values for both the VIC and FAM probes were recorded and the difference between them (ΔCT) was calculated. Samples were analysed in triplicate. Genomic DNA from homozygous individuals, was used as controls to ensure no cross hybridisation occurred between the two probes. The ΔCT of cDNAs was compared with a standard curve of ΔCT values from two homozygous gDNAs mixed at different ratios (49:1, 9:1, 4:1, 7:3, 3:2, 1:1, 2:3, 3:7, 1:4, 9:1, and 1:49), and the percentage expression from each allele was extrapolated. This method was adapted from [45]. The primers used were as follows: MeDIO3UTR-F, 5′CTTCCCTCCTCCCCAAATTCC-3′; MeDIO3UTR-R, 5′-TGCAGTCAACAAAGTGGAGGAA-3′; + allele probe, 5′-(VIC)-TTCTCTCCTTGGTTTTT-(MGB)-3′; and – allele probe, 5′-(FAM)-TTTTTTCTCTCGGTTTTT-(MGB)-3′.
SYBR green qRT-PCR assays.
Assays were performed using the SensiMix NoRef kit (Quantace). The amplification of each primer pair was determined using a serial dilution of cDNA (1, 1/5, 1/25, 1/125, 1/625). Reactions were performed in triplicate, and the average CT value of each dilution was used to generate a standard curve. The slope of the curve when plotted to log10 was used to determine the efficiency of amplification (E) for each primer set using the following equation: E = 10(−1/slope) and the relative fold expression calculation (2– ΔCT) was corrected for the amplification efficiency [46]. The relative gene expression was calculated using the following equation: Expression Ratio = EΔCT(sample)/EΔCT(reference). Sample reactions were performed in triplicate. Three fetal head samples were used (from between d22 and d25 RPY) and three PY brain samples (from between D17 and D20 post partum).
The primers used were as follows: MeDIO3QF, 5′- CCGAGGGCTACAAGATCTCA-3′; MeDIO3QR, 5′- CACGTTTGTTTGGGGTTCTT-3′; MeECR19F, 5′-GCGGCTTCACAAATTTATTTTC-3′; MeECR19R, 5′-CAACTCTGCACAGATGGATGA-3′; MeRan3F, 5′-CAGCTGGATCCAATTTGACA-3′; and MeRan3R, 5′-TTGGACCATGATCCTGGAAT-3′.
Methylation analysis.
Genomic DNA was extracted using standard protocols [47]. 10 μg of restriction enzyme–digested DNA was separated on 0.5× TBE 1% agarose gels and transferred to Hybond-N+ (GE Healthcare) nylon membranes. Filters were pre-hybridised in ULTRAhyb solution (Ambion) at 42 °C for a minimum of 2 h. Probes were labelled with [α-32P]-dCTP using the Megaprime DNA labelling system (GE healthcare) and added to the hybridization buffer. Filters were incubated at 42 °C overnight, washed to the stringency of 2XSSC/0.1%SDS, and exposed to a phosphoimager screen. Probes used for the analysis can be found in Table S3.
Sequence analysis.
With the exception of novel platypus and tammar wallaby sequence generated here, sequences for computational analysis were downloaded from the UCSC genome browser [48]. These sequences are: human sequence, build March 2006, chr14:100210000–101150000; mouse sequence, build February 2006, chr12:109850000–110780000; dog sequence, build May 2005, chr8:71946000–72800000; opossum sequence, build January 2006, chr1:315760000–317570000 (reverse complement); and chicken sequence, build May 2006, chr5: 51365000–51832000.
The repeat content for the Dlk1-Dio3 region in each species was determined using RepeatMasker version: open-3.1.8 [49]. The specific repeat library was used for each species and the default parameters. For the platypus sequence, a command line version of the software was used with a pre-release of the latest library for this species (kindly provided by R. Hubley, RepeatMasker, Institute for Systems Biology, USA).
CpG islands were predicted by CpGplot [50] using the default parameters and a window of 200.
Evolutionary conserved region prediction.
Two different programs were used to predict ECRs: zPicture [50] and mVista [51,52]. The LAGAN algorithm [53] was used in the mVista alignment. The translated anchoring option (where one or more of the alignments steps are performed on translated sequence) was used, because it can improve the alignment of distant homologues. The default setting of >70% identity and >100 bp in length was used between marsupials and platypus. Greater than 55% identity and >80 bp was used for the alignments of eutherians to other species and chicken to other species. For both programs, human, mouse, dog, and chicken sequences were repeat masked using the species-specific setting. Wallaby, opossum, and platypus sequences were first repeat masked with repeats changed to lower case (softmasking).
Each sequence was used in turn as the base sequence for aligning the others to and the coordinates for the same ECR in each species were identified. The coordinates were recorded for the largest region showing homology between the pairwise alignments of any species. Using this approach, the ECR data were merged between the different pairwise alignments generated by each program and between the two programs. In addition, ECRs that were less than 200 bp apart in all species were merged.
Putative miRNAs were identified in using the miR-abela program [29]. Putative CTCF binding sites were identified using the program FUZZNUC to search the forward and reverse sequence of the DLK1-DIO3 region in each species. Three motifs were used CCGCNNGGNGNC [54], CCGCGNGGNGGCAG [55], and CCDSNAGRKGGHDS (which is based on the binding motif identified in a large scale analysis of CTCF binding site in human [56]).
Supporting Information
Figure S1. Overlapping BAC Clones Identified for Sequencing.
(A) Complete coverage of the Dlk1-Dio3 region in Macropus eugenii was achieved through sequencing of BAC clones indicated in red. The thirteen sequenced clones span 1,674,705 bp. BAC details are summarised in Table S1A.
(B) Complete coverage of the Dlk1-Dio3 region in Ornithorhyncus anatinus was achieved through sequencing BAC clones indicated in red. Seven clones spanning 795,237 bp were sequenced. BAC details are summarised in Table S1B.
The Macropus eugenii genomic BAC library was from the Arizona Genomics Institute (average insert size 166 kb) covering 11.36 genome equivalents and cloned into the HindIII site of pCUGIBAC1. The Ornithorhyncus anatinus BAC library was from Clemson University Genomics Institute (average insert size 143 kb) covering 11 genome equivalents. It was constructed in the HindIII site of pCUGIBAC1. Libraries were screened with probes for Dlk1 and Dio3. A Dlk1 probe was generated for wallaby by searching the Monodelphis domestica trace archive with human DLK1 with a probe designed against the most conserved sequence. DLK1 sequence for platypus was identified by searching the platypus trace archive with chicken Dlk1 sequence and a probe was generated to this platypus sequence. Dio3 probes amplified from wallaby and platypus were generated after identification of conserved primers from alignments of human, mouse, rat, and chicken Dio3. BACs identified from library screening were amplified to confirm presence of probe sequences, and HindII digested and fingerprinted allowing them to be aligned and assembled into a contig. One BAC from each end was selected for shotgun sequencing. Using end sequence or fully sequenced BACs, new probes were generated and the process repeated until complete coverage was achieved.
Figure S2. Exon Sequence, Structure, and Polymorphism for Wallaby and Platypus DLK1
(A) mRNA sequence of wallaby and platypus DLK. The protein coding region (CDS) is highlighted turquoise and is in uppercase. The 3′ UTR is highlighted in yellow. The full extent of the 3′ UTR was not established and is illustrated up to a predicted poly adenylation signal (green). All of the splice sites have an intronic GT in the donor site and intronic AG in the acceptor sites (red). The polymorphisms identified within the genes are in bold and surrounded by square parentheses.
(B) Schematic alignment of the intron-exon structure of the gene in human, mouse, wallaby, opossum, platypus, and chicken indicates that the two 3′ marsupial introns are greatly expanded compared to those of the other vertebrates. In eutherians, the Dlk1 gene span is less than 1% of the entire domain (from the start of Dlk1 to the stop codon of Dio3). However, in the noneutherian species analysed, the Dlk1 gene span is greater than 2% of the region.
(C) Sequence traces of genomic DNA and cDNA from heterozygous individuals confirm that the gene is biallelically expressed in wallaby and platypus. Sites of single nucleotide polymorphism are indicated with red arrows.
Figure S3. Wallaby and Platypus DIO3
DIO3 in all vertebrates contains a single exon. The sequence of the DIO3 gene in wallaby and platypus is shown. The coding sequence is highlighted turquoise and is in uppercase. The 3′ UTR is highlighted in yellow. The full extent of the 3′ UTR is illustrated up to a predicted poly adenylation signal (green). The polymorphisms identified within the genes are in bold and surrounded by square parentheses.
Figure S4. Comparative Repeat Content of Regions Showing Expansions/Contractions between Therians
Inter-zone repeat content of regions which show expansions and lack of expansion (contractions) in eutherian and marsupial mammals. LINE elements are subdivided into four families (L1, L2, L3, and RTE) to illustrate the different contributions made to overall LINE content.
Figure S5. Assessment of Noncoding RNA Transcription in Wallaby
(A) Schematic representation of the Dlk1-Dio3 domain indicating locations of ECRs and randomly selected loci assessed for expression. Position of the primer pairs used for the RT-PCR analysis areindicated by black bars below the horizontal line. The positions of primer used to analyse ECR expression are shown in red. Above the horizontal line are ECRs. Turquoise, ECRs conserved in all vertebrates studied; pink, ECRs conserved in all non-eutherians studied; blue, other ECRs. The extent of the expression is indicated by a broken green line.
(B) RT-PCR with primers designed to ECR36 (located within an intron of Mirg in mouse). Expression was seen from wallaby fetal head.
(C) A G/T SNP was identified in one wallaby PY sample. Sequence trace data demonstrate biallelic expression of ECR36.
Figure S6. Wallaby Lacks miRNAs but Has Remnants of RTL1
(A) RTL1 is not expressed in the wallaby. Primers, designed to the reverse transcriptase orthologous region which is expressed in eutherians, failed to amplify cDNA from wallaby placenta, fetal head, or pouch young brain. BYS, bilaminar yolk sac; TYS, trilaminar yolk sac; PY, pouch young.
(B) Sequence alignment of human and mouse miR-127 with the tammar wallaby and opossum miRNAs as predicted bythe miR-abela program [29].
(C) Comparative secondary structure; the known mature miRNA regions are indicated by red parentheses. Orthologous regions to mature miRNAs are indicated by blue parentheses
(D) The pre-RNA region was not amplified from wallaby pouch young brain cDNAs.
Table S1. Wallaby and Platypus Sequenced Clones
(A) List of sequenced wallaby BACs including accession numbers and finished lengths. (B) List of sequenced platypus BACs including accession numbers and finished lengths.
Table S2. The ECRs Identified in the Dlk1-Dio3 Region in Human, Mouse, Dog, Wallaby, Opossum, Platypus, and Chicken
Each ECR is give a number from 1 to 141. In addition, the 31 vertebrate ECRs are indicated by V1 to V31. The coordinates of each ECR are given for their position in the analysed sequences. ECRs, which are inverted in at least one species are indicated by (–). One ECR (ECR64) is duplicated in the human sequence.
Table S3. Summary Expression Analysis Indicating Absence or Presence of Expression
Table S4. List of PCR Primers Used for Expression Analysis and Southern Hybridisation Probes
The PCR conditions used are indicated.
Table S5. List of Wallaby Samples Used in This Analysis
By manipulating the reproductive cycle of the tammar wallaby, it is possible to recover animals at specific stages of development. This is because lactating mothers have a second fertilised embryo arrested in diapause. During the wallaby breeding season, pregnancy can be initiated by the removal of the pouch young (RPY), which reactivates the blastocyst from diapause. Pregnancies are dated relative to the day of pouch young removal and are prefixed with d (e.g., d23). Pouch young are dated from days post parturition and are prefixed by a D (e.g., D10).
We are grateful to Wolf Reik, Gavin Kelsey and Malcolm Ferguson-Smith for helpful discussions. We thank R. Hubley for providing the repeat-masked platypus sequences prior to release. We also thank Marika Charalambous, Rachel Jackson, Mitsuteru Ito, Sue Osborne, Nadine Richings, Herve Seitz, and Eleanor Ager for technical and intellectual input during the course of the work. Information about the SAVOIR consortium and its participants can be found at http://www.sanger.ac.uk/PostGenomics/epicomp/. The Savoir Consortium (in addition to named co-authors) are: Sanger Institute, Hinxton Cambridge: Alex Bateman, Chao-Kung Chen, John Collins, James Gilbert, Elizabeth Huckle, Sam Griffith-Jones, Jennifer Harrow, Matthew Jones, Mustapha Larbaoui, Karen Oliver , Carol Scott, Sarah Sims, Charles Steward, and Jennie Yang. Babraham Institute, Cambridge: Guillaume Smits, Simon Andrews, Delphine Beury, Christel Krueger, Elena Ivanova, Iain McKendrick, Paul Smith, Gavin Kelsey, and Wolf Reik.
Author Contributions
CAE, AJM, ID, MBR, and ACF-S conceived and designed the experiments. CAE, AJM, LM, DJG, and AJP performed the experiments. CAE, AJM, LM, ER, and ACF-S analyzed the data. ER, AJP, GS, JAMG, JR, ID, and MBR contributed reagents/materials/analysis tools. CAE and ACF-S wrote the paper.
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subset: commoncrawl


text:
Required Textbook:
Aging Matters
Edition: 1st*
The text may be purchased at the SCC Campus Bookstore, or Student Book Exchange, or various online bookstores like Amazon or Half.com; or download an e-text, or check into renting a book at Chegg.com.
There should also be a copy of the text at the SCC Main Campus Library Reserve Desk, Davis Center, and West Sacramento Center for a 2 hour maximum checkout.
*If you choose to use a different edition of the text, it is your responsibility to compare your text with the current text for updated information and reorganization of the material. (Older copies of the text might be found on the 3rd floor of the Learning Resource Center.)
Video Series Enhanced Online-Class
Growing Old in a New Age
The VIDEO SERIES "Growing Old in a New Age" accompanies this course. You will view 13 one-hour programs introducing key concepts and facts. Selected experts provide commentary.
Where to View Course Videos: The video series may be viewed at the Annenberg Learner web site.


text:
I'm a professional PHP programmer with almost a decade of professional experience and considerably more as a hobbyist programmer. I've also had some exposure to Java, Object Pascal and am interested in learning objective C
I've started work on a PHP framework, though it's still at a very early stage and not yet really useful. The current source is available on GitHub.
I've also begun development of a CSS3 elastic grid for use on my own projects and had published that on GitHub as well.
2 answers
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text:
my hard drive was filling up so i used a small app called SpaceSniffer to see what was taking up the space and the above image is what it found. what are the...
Type: Posts; User: mikbhoy; Keyword(s):
my hard drive was filling up so i used a small app called SpaceSniffer to see what was taking up the space and the above image is what it found. what are the...
Python also installs with the game Krater.
sorry been busy at work, almost forgot all about this question
OS - Windows 7 64 bit SP1, all up to date.
router - BT Homehub 2, all up to date.
latest version of steam. (not the beta one)
hi, hope this is in the correct section.
ive been playing Skyrim a lot lately through Steam and after i stop playing i cant connect to the internet (constant time outs), i usually have to restart...
ye i thought as much mate, cheers.
btw as i said i use eset :p
sure can
i used the link myself and got no such warning, im not using bullguard though i use eset smart security.
hi guys, i posted a link to the non destructive win 7 reformat article on a forum i go to and when one of the guys clicked the link he got this message from Bullguard
'Originally Posted by...
eset smart security also goes nuts about this download.
i think he may have been talking about this site http://ninite.com/
Review on Techspots
the spiel
Sharing your Wi-Fi connection with Connectify is pretty straightforward. Once installed, simply create an access point name, give it a WPA2 encrypted passphrase...
thanks for this, as of right now its still running and as they are having probs replying to all the emails they have recieved you no longer need to fill out any forms, you can just dl the full free...
how about adding a thanks button to the bottom of posts so users can thank helpfull people without having to make another post just to say thanks.
first up
IOBit Smart Defrag from here http://www.iobit.com/iobitsmartdefrag.html , its free and goes beyong just defragging as it also has disk optimization options. im using this just now and...