text: On 02/01/2009 09:13 AM, Gaal Yahas wrote: > #2 is very near what you get today by looking at source code in an > environment that attempts to run the Unicode BiDi algorithm on your > source, for example a browser. In my experience, this can be very > confusing. > > If I'm understanding your proposal correctly, in #2 you still want to > fix the crazy cases of flipped capture parentheses etc., but that > implies your editor must understand regular expressions, which at best > it will do when something is a regexp literal but which will fail when > the programmer is constructing a regexp piecemeal from strings. > Yes, thats why I said "ideal editor" - assume for the purpose of discussion that it can magically distinguish strings literals which are used for RE from general purpose strings (which are treated as tokens and have the general bidi algorithm applied to them). I imagine a practical solution would allow the user to manually adjust "roles" for literal strings (however, I do not know where it would save this info for future edits...). If you construct your regexp from pieces, I expect each part to be meaningful as re on its own, so have the same rules. > Indeed, your solution doesn't attempt to handle simple strings, so the > user can get confused by inconsistency issues: why $foo = "SHALOM" but > $foo = qr/MOLAHS/? Actually, the suggestion for source code (programming languages dependant - again we assume full syntax awareness) specifically defines string literals and comments as tokens, so it would be $foo = "MOLAHS" as well. > Also, there's the tokenization problem you already > mention: NATBA"G would come out very bad from this transformation. > In #1 certainly it would not be much different than NATBAG. In #2, since " is not a special character in RE, it will be included in the token and have the standard Bidi algorithm applied to it - so you'd get G"ABTAN , just as you'd probably expect. > So if these are the two options, I'd vote for #1. (I'd also recommend > in source code representing invisible override characters as entities, > so that if the actual data has RLM/LRM/etc. marks, where possible it's > better not to have those as literals in the source code. That's a > recommendation for the end programmer, not the editor, I think.) > Another one would probably be to use i18n and have all user-destinated strings in po files, which as a nice byproduct would also reduce potential problems with string literals... > Perhaps one day we can have an alternate surface syntax for strings > and regular expressions that is designed to be RTL friendly. Hebrew or > Arabic metacharacters, introduced with slashes instead of backslashes. > This would be used in conjunction with an editor hint that makes the > whole expression RTL (in Unicode terms, sets the paragraph > directionality). If used on whole lines of code, it can also change > the alignment to be right-justified. Perl 6 has many quotelike > operators, with room for extensions like this. The problem with these > things is that they're hard to get right, and pesky problems will > still appear no matter how hard you try if the data really does > contain mixed directionality characters. We can ask Larry for his > opinion, though. > Interesting idea. We tend to think in terms of adjusting bidi display algorithms to cope with existing syntax. Adjusting syntax to cope with bidi issues would probably be less quirky and easier for editor devs to implement. Thanks for this input, Amit > On Sun, Feb 1, 2009 at 5:08 AM, Amit Aronovitch <aronovitch at gmail.com> wrote: > >> Hi, >> >> Following a discussion I took part in about standartization of the >> display of Hebrew text in structured expressions and source code, I >> would be happy to hear some opinions about how we would like regular >> expressions containing bidi chars to be displayed (in an "ideal >> editor" that is fully syntax aware). >> >> In the examples below, caps represent RTL characters and lowercase LTR chars. >> >> The basic principle that was proposed (for structured expressions) is >> that text should be split into "separators" and "tokens" according to >> the relevant syntax, the general-purpose Bidi rules be applied within >> each token only, and then tokens and separators should be concatenated >> left to right always. >> >> Applied to regular expressions, I thought that since in RE each >> pattern character is an atom (1), then this effectively means to force >> LTR everywhere (except maybe stuff like named captures >> (?<NAME>...) etc.). >> However, it was suggested instead that any sequence of pattern >> characters (not containing "special" characters) should be treated as >> a token (2). >> This would make simple searches easier to read, >> >> e.g. /SHALOM/ would be displayed /SHALOM/ by (1), >> but /MOLAHS/ (much more readable if it was actual Hebrew) by (2). >> On the other hand, /YADAII?M/ in (2) would show as /IIADAY?M/ , >> which is very confusing, so I thought the simplification was not worth it. >> >> However, I am used to languages where simple searches are commonly >> done by other means, whereas in Perl using RE for simple text search >> might be more common because of the specialized syntax. What do you >> think? >> >> Amit >>


text:
Eric attended the Honors College at Arizona State University. After receiving a BA in Theater, he and his wife Laura set off for New York City. There, Eric earned a degree in illustration from Pratt Institute and Laura decided to pursue a career in medicine. Soon after, Pattern stole Eric away from his freelance jobs with nationally distributed magazines such as Culture and Alaska Magazine. At Pattern, he excels at converting ugly, confusing PowerPoint diagrams into communicative works of art and expertly illustrates almost anything our clients can dream up.
Eric's independent illustrations, and information on how to hire him for illustration work, can be found at his website, www.ericpiatkowski.com.
little known fact: Eric bikes to work almost every day in the summer.

subset: commoncrawl


text:
This antique bronze compass design was originally commissioned by England's King George IV.
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text:
Syracuse, NY -- A rash of break-ins around Syracuse's Salt Springs neighborhood and nearby Erie Boulevard East continued this year's trend of more burglaries compared to a year ago.
According to the Onondaga County crime database, there were 65 burglaries from Nov. 12 to 18, compared to 46 the same week a year ago.
So far this year, there have been 2,476 burglaries across Onondaga County, compared to 2,145 for the same period a year ago, the database showed. That's a 15 percent increase.
In Syracuse, there have been 1,620 burglaries so far this year, compared to 1,461 a year ago. The city recorded 65 percent of the burglaries countywide. But the burglary rate in the suburbs went up slightly more than in the city this year. Last year, the city accounted for 68 percent of the burglaries.
In the most recent week, Nov. 12 to 18, there were four burglaries in Salt Springs and three more along the Erie Boulevard East commercial district nearby.
The city's North Side neighborhood bounded by Butternut, Court and Lodi streets and Grant Avenue was also hit hard, with 11 burglaries in that dense residential area.
Three burglaries were also reported in a small area around Ballantyne Road west of Onondaga Creek in the North Valley neighborhood.
Overall, there were 204 crimes the week of Nov. 12 to 18 compared to 210 crimes the week before.
The database includes a record of aggravated assaults, arsons, burglaries, all larcenies, murders or attempted murders, robberies and vehicle thefts. These crimes are defined as Part 1 offenses by the FBI. Here is a list of New York State crimes that are included in these categories.
Crimes investigated by state police are not included in this database. Their territory includes part of southern Onondaga County. Troopers operate on a different computer system that crosses county lines. At this time, that system is not compatible with the database used by the rest of the county.
The Reported Crimes database does not include every serious crime, but provides a snapshot of crime in the Syracuse area. Many departments take several days to several weeks to compile their reports. The crime statistics are updated weekly back to Jan. 1 of this year.
Read more:
» See a snapshot of Syracuse crime data from 2010 and 2011.
» Check your own neighborhood in the Reported Crimes database.
»: See a list of all crimes listed in the Onondaga County Reported Crimes Database, listed by type.
» Read more about what you'll find in the Reported Crimes database.
» Check The Post-Standard's Police Blotter for a list of arrests.
» Read all of our stories about the Reported Crimes database.


text:
Chips Ahoy! are known for producing crunchy chocolate chip cookies with a vaguely pirate-y name, but at the end of February, they'll release a line of four flavors of "Ice Cream Creations" -- Dulce de Leche, Mint Chocolate Chip, Mocha Chunk, and Root Beer Float. No freezer required.
Find out if they're worth your hard-earned scratch:


text:
Norelco PC60
Ikegami HK312
Sony BVP-360
NBC Chimes
TV News Story
The Ellerbee Collection
Bobby Ellerbee has been collecting vintage television broadcast
cameras for only about two years now.
In less than one year though, he was able to put together a
beautiful nine camera collection, and he is fast becoming one
of the leading experts on vintage TV cameras and their history,
in North America.
Bobby has done extensive research on the history surrounding
each of his cameras and it is in that activity and those whom
he has contacted that has led him to create the "Ellerbee
Classic Camera Survey."
It is a first-ever, comprehensive tally and listing which he
has created that makes a valiant effort to count up how many
of these valuable and often-times historic vintage cameras have
actually survived, and where they are.
His efforts have spanned to searching collections from The
Smithsonian Institute, university archives, most of the major
broadcast museums and numerous private individuals. His scholarship
provides a one-of-a-kind list of many the rare vintage cameras.
His diligent efforts are sincerely appreciated by all of us
who have a passion for television and it's contributions to
our Society.
Displaying his a truly generous spirit, Bobby has played a major
role in locating many important artifacts of video history for
The Museum of Broadcast Technology near Boston,Massachusetts.
Bobby has used his talents as both a detective who could rival
Sherlock Holmes, as well as his powers of articulate persuasion
to build collegial relationships between potential donors or
providers of historic items and the folks at The Museum of Broadcast
Bobby has been a key player in successful acquisitions of rare
RCA TRT-1A/B 2" Quad machines, as well as RCA TR-22 and TR-600
quad video tape equipment. The MBT's talented Tom Sprague has
brought one of those TR-22s back to life ! The TR-600s are next
Most recently, thanks to Bobby's recent leadership direction
and assistance in the MBT's acquisition of an historic RCA TK-40A
Image Orthicon color camera. This camera is one of a pair delivered
in 1954 to what was then WKY-TV in Oklahoma City, OK. These
were the first two color TV cameras ever delivered to a local
independent commercial TV station in the world. Fortunately
the other TK-40A is at the Oklahoma Historical Center.
The Museum of Broadcast Technology is eternally grateful to
Bobby for his passion, dedication and to the preservation of
vintage cameras and related broadcasting artifacts. Way-To-Go
Bobby !
Most sincerely,
Paul R. Beck, President,
Thomas R. Sprague, CFO,
Dr. Henry Berman,
Museum of Broadcast Technology, Inc. www.wmbt.com
How My Collection Got Started
Around age 10, (1960), I was totally hooked on drawing
television cameras and I drew them all day long for many years.
My favorite was the TK41 (second favorite was TK11 then Norelco)
and a lady named Kathryn S. Cole at NBC's 30 Rockefeller Plaza
kept me loaded with thick stacks of 8x10 glossy back stage photos
for many years. One of them is on our home page and others are
scattered around, but all are on the RCA
TK 40-41 picture pages.
The RCA TK44B my first camera, is the start of a very
interesting story. Wanting my own TV camera, I called Chuck
Pharis in California on August 8, 2006 to ask the best way to
go about this. Later that day, after a good talk with Chuck,
I decided to just call around here in my area (Athens Atlanta)
and just ask if anyone had any old cameras they would like to
donate to a collector. On my first call, I left a message which
I usually never do, and then got busy with other things and
made no more calls.
Later that night, an engineer at the station, where
I had left the message, Gary Coffman, WXIA-TV in Atlanta, called
and asked me if I would like to have 7 cameras, a TD8 HF pedestal
and 3 miles of cable.
There were 5 RCA TK44Bs and 3 RCA TKP 45 portable cameras
and 8 CCUs. I kept 3 TK44s. I gave Chuck 2 of the 45Ps and gave
Paul Beck and Tom Sprague 2 TK44s, the cable, the CCUs and a
the other TKP-45 for their Museum of Broadcast Technology that
is being built in Woonsocket Rhode Island, just outside Boston.
Tom and Paul gave me a CBS Philips PC60, 1 Vinten head
from WGBH (Boston) and a tripod that was used by WPIX only at
Yankee Stadium (outfield camera). The TK-44Bs came from Dixie
Sports Net, an Atlanta production company that went out of business,
but, these all these cameras actually started service at NBC's
Burbank Studios.
How do we know? The NBC Burbank cameras have had an
extra exhaust fan added to the top of the camera. This was a
modification made by necessity because the viewfinders kept
overheating and they would actually black out. This was not
so much a problem at local stations because their cameras did
not work as many hours as the NBC Burbank cameras.
If you go through the TK
44 "At Work" picture pages on this site, or the
Old Radio RCA Equipment website, and look at all the TK44s,
aside from the NBC network TK44s, there is only one other TK44
picture that has the extra fan mod on top and that was from
Oral Roberts University, or O.R.U.
Roberts did a lot of taping at the NBC Burbank facility
and when NBC sold some TK44s and TK45Ps (and they were known
to have bought and sold a lot of TK44s), I believe that all
these cameras went to Oral Roberts University. There are O.
R. U.property tags on the lenses.
As for the TK45Ps, I think these were originally used
on Saturday Night Live and these NBC New York cameras went to
Burbank, then to O.R.U., then Dixie Sports Net, then Gary Coffman,
then to me, Chuck and MBT (abbreviation for Museum of Broadcast
Home / Privacy / Legal / Links / Contact
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text:
The road heading north from Mytilíni towards Mandamádhos follows a rather nondescript coastline, but offers startling views across the straits to Turkey. South of the capital, there are two fine museums at Variá and a couple of decent beaches further down the peninsula.
Greece // The East and North Aegean //


text:
WATCH: The New Slightly Naughty Magic Mike Trailer
At this rate we won't have to see Magic Mike—Warner Bros is teasing out the whole thing in the trailers and stills the studio is releasing. Then again, we want to see Matt Bomer's bare tush as large as possible—so we hope they show it in IMAX 3-D.
By the way, while we appreciate the eye candy, you have to kind of laugh at how unerotic strippers are in female-orientated clubs. They have to be in some ridiculous costume and always end up doing pushups or rodeo movies. We once saw a go-go boy in Chicago open a beer bottle with his butt. Now that's hot!
Matt Bomer: Gorgeous-check, Talent-check, Role Model-check, Classy-check, Hot Bum-check and double check
@Nigel: check you out.
Oh my God.
I might actually see this now.
this looks so terrible
Just how much is Queerty getting paid to promote this movie? I noticed how since it shows Matt Bomer's ass they're not going on about how (in queerty's opinion) he's still somehow closeted even though he's been out for awhile now like they have done in previous recent article about Bomer.
@Adam: Apparently you have not heard of the site cam4. You will see a lot more there, online, and free. Or go spend your $$$ to get a few half second flashes of ass and faux dick. But do check back and share what it was like being the only dude, albeit gay, in a theatre full of horny cougars, and the Queerty staff.
Oh the poor, poor ushers. The inside of those menopausal housewife infested theaters is going to smell worse than a six week-old fish market in the middle of July.
Thankfully we know that at least one of the guys on stage is family.
@JayKay: Thank you for confirming that you'll be watching the film… numerous times. :) Don't let the bashers catch you. We wouldn't miss you if you're gone but I'm sure someone self-deprecating will. :)
Simon Halls, the luckiest man in Hollywood.
JayKay-And I'm sure you'll be on your knees servicing each and every woman there and licking their clits, labia, and tongue fucking them! ;)
Too perfect to be real. I bet Bomer has few skeletons in his closet.
@Ted B. (Charging Rhino): You mean luckiest man on planet earth
just so there's no confusion, this opens in the US on friday, june 29 (it's in the UK on july 11).
It does look a bit naff, even for a Steven Soderbergh film.
I wouldn't bother going to see a film specifically to see Channing Tatum or Matthew M. or whoever else is in it, but I might go and see it just for Matt Bomer…
@JayKay: Misogynize much? (and yes, I know "misogynize" is not a real verb).
@Adam: lol ditto
So far I've only seen them getting half naked, but what is the story of the movie??? If it has one…
@Ru…: who cares?? you stfu when channing tatum is getting naked!
Is this produced by Lifetime channel for women? This has to be marketed to suburban white housewives …and of course there is no men of color! Typical Queerty, hustling some faux "gay" nonsense.
The str8 strippers I know dance for guys. But that wouldn't sell to Middle America. Wonder if the film will feature any gay characters (esp. self-respecting gay characters in the film who aren't cartoons), as that is obviously part of their intended audience. Or will the movie pretend we don't exist.
Channing and Alex make me kahraazzee…but i'll wait 4da directors cut version online or dvd
if there is no age restriction on this i may just watch it XD
Still not sure I care.
@Matt: u cant just sit there and enjoy the guys? hv a drink…or two…bottles


text:
The National Weather Service has issued a tornado watch for all of southeastern Louisiana and southwestern Mississippi until 11:00 a.m. Tornado warnings issued earlier for Tangipahoa, St. Tammany and Washington parishes in Louisiana and Pearl River County in Mississippi expired at 7:00 a.m.
A severe thunderstorm warning for the south shore expired at 7:45 a.m.
Forecasters say showers and thunderstorms are likely throughout the morning, and there's a chance of showers in the afternoon. Some thunderstorms may be severe.
Highs will be in the lower 60s, with northwest winds at 10 to 15 mph. The chance of rain is 80 percent.
Tonight is expected to be much colder, with lows in the mid 30s. It will be a clear night with northwest winds at 10 to 15 mph.
Today also heralds the end of the Atlantic hurricane season. For a look back at the 2010 storms, from Alex to Tomas, see www.nola.com/hurricane.


text:
Tuesday, April 24, 2007
2 Comments:
- Pheealzabub said...
No problem... We all need to deal with life when it rears its ugly head. Looking forward to your thoughts on the Tillman/Lynch testimony from Tuesday (no pressure or anything...)
- 阿 said...
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text:
I've noticed that my caffeine effect (mild shakiness) are nil when I moderate the coffee with cocoanut milk (lots of it). I used to love lattes in my dairy days. But even with the milk I'd have nasty effects with the second cup. Now I can drink two full mugs without feeling anything like jitters. But only when I really bump it up with that delicious, fatty cocoanut milk.
Good to know that coffee can be so powerful.


text:
Patent application title: Thermostable Nucleic Acid Polymerase From Thermococcus Gorgonarius
Waltraud Ankenbauer (Penzberg, DE)
Vitaly Svetlichny (Bayreuth, DE)
Elizaveta Bonch-Osmolovskaya (Moscow, RU)
Christine Ebenbichler (Antdorf, DE)
Bernhard Angerer (Rosenheim, DE)
Gudrun Schmitz-Agheguian (Bernried, DE)
Frank Laue (Paehl-Fischen, DE)
Roche Diagnostics GmBH
IPC8 Class: AC12N912FI
USPC Class:
Class name: Enzyme (e.g., ligases (6. ), etc.), proenzyme; compositions thereof; process for preparing, activating, inhibiting, separating, or purifying enzymes transferase other than ribonuclease (2.) transferring phosphorus containing group (e.g., kineases, etc.(2.7))
Publication date: 2011-01-27
Patent application number: 20110020898
Sign up to receive free email alerts when patent applications with chosen keywords are published SIGN UP
A purified thermostable enzyme is derived form the thermophilic
archaebacterium Thermococcus gorgonarius. The enzyme can be native or
recombinant, retains approximately 90% of its activity after incubation
for two hours at 95° C. in the presence of stabilizing agents and
possesses 3'-5' proofreading exonuclease activity. Thermostable DNA
polymerases are useful in many recombinant DNA techniques, especially
nucleic acid amplification by the polymerase chain reaction (PCR).
1. A process for preparation of a DNA polymerase, the process
comprising:growing a microbial host cell comprising a DNA encoding a
thermostable DNA polymerase obtainable from Thermococcus gorgonarius
which catalyses the template directed polymerization of DNA, possesses
3''-5''-exonuclease (proofreading) activity and is characterized by at
least a two-fold greater replication fidelity than DNA polymerase
obtainable from Pyrococcus furiosus, whereinthe DNA polymerase comprises
the amino acid sequence set forth in SEQ ID NO:7; orthe DNA comprises the
nucleotide sequence set forth in SEQ ID NO:6; orthe DNA polymerase has an
apparent molecular weight between about 92,000 and 96,000 daltons;
andpurifying the DNA polymerase from the host cell.
2. The process of claim 1, wherein the host cell comprises a vector and said vector comprises the DNA encoding a thermostable DNA polymerase.
3. The process of claim 1, wherein the DNA polymerase comprises a coding sequence for the DNA polymerase, amplifiable from T. gorgonarius using primers consisting of SEQ ID NO:4 and SEQ ID NO:5.
4. The process of claim 1, wherein the DNA polymerase comprises the amino acid sequence set forth in SEQ ID NO:7.
5. The process of claim 1, wherein the DNA comprises the nucleotide sequence set forth in SEQ ID NO:6.
6. The process of claim 1, wherein the DNA polymerase has an apparent molecular weight between about 92,000 and 96,000 daltons.
7. The process of claim 1, wherein the host cell is E. coli.
[0001]The present patent application is a divisional application of 12/135,994, filed Jun. 9, 2008, which is a divisional application of U.S. patent application Ser. No. 09/269,860, filed Apr. 1, 1999, now U.S. Pat. No. 7,425,423, issued Sep. 16, 2008, which is a US national phase of PCT/EP97/05393 (later published as WO 98/14590), filed Oct. 1, 1997, which claims priority to European Patent Application Nos. 96115874.8, filed Oct. 3, 1996 and 97100584.8, filed Jan. 16, 1997, each of which is incorporated by reference.
[0002]The present invention relates to an extremely thermostable enzyme. More specifically, it relates to a thermostable DNA polymerase obtainable from Thermococcus gorgonarius.
[0003]DNA polymerases are a family of enzymes which are in particular involved in DNA replication and repair. Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli (see, e.g., Bessman et al., J. Biol. Chem., 223:171-177 (1957), and Buttin and Komberg, J. Biol. Chem., 241:5419-5427 (1966)).
[0004]Research has also been conducted on the isolation and purification of DNA polymerases from thermophiles, such as Thermus aquaticus (Chien, A. et al., J. Bacteriol., 127:1550-1557 (1976)). Further, the isolation and purification of a DNA polymerase with a temperature optimum of 80° C. from Thermus aquaticus YT1 strain has been described (EP 0 258 017 and U.S. Pat. No. 4,889,819).
[0005]Research has indicated that while the Taq DNA polymerase has a 5'-3' polymerase-dependent exonuclease function, the Taq DNA polymerase does not possess a 3'-5' proofreading exonuclease function (Lawyer, F. C. et al., J. Biol. Chem., 264:6427-6437 (1989); Bernad, A. et al., Cell, 59:219 (1989)). As a result, Taq DNA polymerase is prone to base incorporation errors, making its use in certain applications undesirable. For example, attempting to clone an amplified gene is problematic since any one copy of the gene may contain an error due to a random misincorporation event. Depending on where in the PCR cycle that error occurs (e.g., in an early replication cycle), the entire DNA amplified could contain the erroneously incorporated base, thus, giving rise to a mutated gene product. Furthermore, research has indicated that Taq DNA polymerase has a thermal stability of not more than several minutes at 100° C.
[0006]The 3'-5' exonuclease activity is generally considered to be desirable, because misincorporated or unmatched bases of the synthesized nucleic acid sequence are eliminated by this activity. Therefore, the fidelity of PCR utilizing a polymerase with 3'-5' exonuclease activity is increased. Such an enzyme is, e.g., the DNA polymerase from Pyrococcus furiosus (Lundberg et al., Gene, 108:1-6 (1991)).
[0007]Other more recent investigation focuses on the isolation and purification of DNA polymerases from archaebacteria such as Thermococcus sp. (EP 0 455 430), in particular a purified DNA polymerase obtainable from Thermococcus litoralis is described. Also the recombinant preparation and the gene encoding for this enzyme is known in the art (EP 0 547 920).
[0008]In EP 0 455 430 is also described a DNA polymerase from Pyrococcus sp. and the gene thereof which also contains introns to be removed for expression of the functional enzyme in E. coli.
[0009]In EP 0 701 000 A and in Proc. Natl. Acad. Sci. USA, Vol. 93, No. 11, pp. 5281-5285 (1996), a thermostable DNA polymerase 9° N7 is described which exhibits a very strong 3'-5'-exonuclease activity. However, it has been observed that the 9° N7 polymerase exhibits a tendency to degrade single stranded DNA (primer). Therefore, the exonuclease activity has been modulated and a mutant 9° Nm polymerase has been obtained which is more useful for a number of applications as the native enzyme. However, when using a 9° Nm polymerase for PCR (see FIG. 6), a primer-template independent DNA-synthesis seems to occur (as can be deducted from the observed highmolecular smear in the gel (FIG. 6)) instead of the occurrence of defined PCR products when using, e.g., Taq-Polymerase. Therefore, neither the native nor the exonuclease modulated 9° N7 polymerase can be successfully used in PCR.
[0010]In WO 92/03556, a thermostable DNA polymerase obtainable from the eubacterium Thermotoga maritima is described which also exhibits proofreading activity. However, in comparison to other DNA polymerases, e.g., Pfu polymerase or Tgo polymerase, the Tma polymermase exhibits a relatively low fidelity (Flaman, J. M. et al., Nucl. Acids. Res., 22:3259-3260 (1994); Cline, J. et al., Nucl. Acids. Res., 24:3546-3551 (1996)).
[0011]The DNA polymerase obtainable from Pyrococcus furiosis (Pfu) is described in WO 92/09689 and exhibits a relatively high fidelity.
[0012]Accordingly, there is a desire in the art to obtain and produce a purified, highly thermostable DNA polymerase with 3'-5' proofreading exonuclease activity which exhibits a high fidelity and is suitable to improve the PCR process.
[0013]The present invention meets this need by providing a DNA polymerase from Thermococcus gorgonarius (Tgo), together with the related DNA and amino acid sequence information, recombinant expression vector and a purification protocol for said DNA polymerase. The DNA polymerase according to the present invention exhibits more than a two fold greater replication fidelity than known DNA polymerases, e.g., obtainable from Pyrococcus furiosus. A further advantage is that the 3'-5' exonuclease activity found in T. gorgonarius polymerase can also decrease non-specific background amplification in PCR by degrading defrayed ends of primers bound to unspecific sequences thereby destabilizing the binding of the primer because of decreasing the length of the helix. Tgo polymerase is thus unexpectedly superior to known DNA polymerases in amplification protocols requiring high fidelity DNA synthesis (see FIGS. 8-10). Another advantageous property of the DNA polymerase of Thermococcus gorgonarius is the fact, that the gene does not contain intervening sequences which would have to be removed to accomplish expression in E. coli.
[0014]The thermostable DNA polymerase enzyme obtainable from T. gorgonarius catalyzes the template directed polymerization of DNA, has an apparent molecular weight of about 92,000-96,000 daltons and retains 90% of its activity after incubation for two hours at 95° C. in the presence of a stabilizer like a non-ionic detergent as, e.g., 0.01% THESIT® (Dodecylpoly(ethylenglycolether)n) or 0.01% NONIDET P40® (Ethylphenolpoly(ethylenglycolether)n).
[0015]Moreover, DNA encoding the 92,000-96,000 daltons thermostable DNA polymerase obtainable from Thermococcus gorgonarius has been isolated and which allows to obtain the thermostable enzyme of the present invention by expression in E. coli. The DNA sequence of the DNA polymerase obtainable from Thermococcus gorgonarius is shown in SEQ ID NO:6.
[0016]The recombinant Thermococcus gorgonarius DNA polymerase also possesses 3'-5' exonuclease (proofreading) activity. Furthermore the gene encoding DNA polymerase from Thermococcus gorgonarius does not contain intervening sequences.
[0017]Thermococcus gorgonarius was isolated from E. A. Bonch-Osmolovskaya and V. A. Svetlichny, Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia. Thermococcus gorgonarius is a new strain, isolated from a thermal vent in New Zealand. This strain does not show DNA-DNA homology with T. celer, T. litoralis or T. stetteri (E. A. Bonch-Osmolovskaya, unpublished results).
[0018]The preferred thermostable enzyme herein is a DNA polymerase obtainable from Thermococcus gorgonarius DSM 8976 (deposited on the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig). This organism is an extremely thermophilic, sulfur metabolizing, archaebacterium, with a growth range between 55° C. and 98° C.
[0019]A preferred method for isolation and purification of the enzyme is accomplished--after all growth--using the multi-step process as follows:
[0020]First, the frozen cells are thawed, suspended in a suitable buffer such as buffer A (40 mM Tris-HCl buffer, pH 7.4; 0.1 mM EDTA, 7 mM 2-mercaptoethanol; 1 mM Pefabloc SC® (4-(2-Aminoethyl)-benzolsulfonylfluorid)), disrupted by high pressure at 1.200 bar. KCl was added to the extract to a final concentration of 400 mM and the solution cleared by centrifugation. The supernatant is then passed through a Heparin Sepharose Cl 6B column (Pharmacia), which has a strong affinity for nucleic acid binding proteins. The nucleic acids present in the supernatant solution of Thermococcus gorgonarius and many of the other proteins pass through the column and are removed by washing the column with two column volumes of buffer A. After washing, the enzyme is eluted with a linear gradient from 0 to 1 M NaCl in buffer A. The peak DNA polymerase activity is dialyzed and applied to a DEAE Sephacel column (Pharmacia). The column is washed with buffer A and the enzyme activity eluted with a linear gradient from 0 to 1 M NaCl in buffer A. The peak DNA polymerase activity is dialyzed and applied to a Cellulose Phosphate column (Whatman). The enzyme is again eluted with a linear gradient such as 0 to 1 M NaCl in buffer A. The enzyme is about 40% pure at this stage.
[0021]The apparent molecular weight of the DNA polymerase obtainable from Thermococcus gorgonarius is between about 92,000 to 96,000 daltons when compared with DNA polymerases of known molecular weight, such as E. coli DNA polymerase I and Thermus thermophilus DNA polymerase. It should be understood, however, that as a protein from an extreme thermophile, Thermococcus gorgonarius DNA polymerase may migrate during electrophoresis at an aberrant relative molecular weight due to failure to completely denature or other intrinsic properties. The exact molecular weight of the thermostable enzyme of the present invention may be determined from the coding sequence of the Thermococcus gorgonarius DNA polymerase gene. The molecular weight of the DNA polymerise may be determined by any technique, for example, by in situ analysis after separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described in Spanos, A. and Hubscher, U., Methods in Enzymology, 91:263-277 (1983).
[0022]Polymerase activity is either measured by the incorporation of radioactively labeled deoxynucleotides into DNAse-treated, or activated DNA, following subsequent separation of the unincorporated deoxynucleotides from the DNA substrate. Polymerase activity is proportional to the amount of radioactivity in the acid-insoluble fraction comprising the DNA, as described by Lehman, I. R. et al., J. Biol. Chem., 233:163 (1958), or by incorporation of digoxigeninlabeled dUTP and determination of incorporated Digoxigenin-dUTP using chemoluminescence according to the method described in Holtke, H. J. et al., Biotechniques, 12:104-113 (1992).
[0023]The DNA polymerase of the present invention has a very high thermal stability at 95° C. It retains approximately 90 percent of its activity after incubation at 95° C. for 120 minutes in the presence of stabilizer. The thermal stability is determined by pre-incubating the enzyme at the temperature of interest in the presence of all assay components (buffer, MgCl2, deoxynucleotides, activated DNA and a stabilizer like 0.01% THESIT® and 0.01% NONIDET P40®), except the single radioactively-labeled deoxynucleotide. At predetermined time intervals, ranging from 1-120 minutes, small aliquots are removed, and assayed for polymerase activity using one of the methods described above.
[0024]The thermostable enzyme of this invention may also be produced by recombinant DNA techniques, as the gene encoding this enzyme has been cloned from Thermococcus gorgonarius genomic DNA. The complete coding sequence for the Thermococcus gorgonarius DNA polymerase can be derived from the plasmid pBTac2Tgo on an approximately 2.3 kB EcoRI/PstI restriction fragment.
[0025]The production of a recombinant form of Thermococcus gorgonarius DNA polymerase generally includes the following steps: DNA is isolated which codes for the active form of the polymerase. This can be accomplished, e.g., by screening of a DNA library derived from the genomic DNA of T. gorgonarius using the DNA sequence described in SEQ ID NO:1 as a probe. Clones containing DNA fragments of T. gorgonarius hybridizing to the probe are isolated and the nucleotide sequence of the plasmid inserts determined. Complete isolation of the coding region and the flanking sequences of the DNA polymerase gene can be performed by restriction fragmentation of the T. gorgonarius DNA with another restriction enzyme as in the first round of screening and by inverse PCR (Innis et al., PCR Protocols, Academic Press, Inc., 219-227 (1990)). This can be accomplished with synthesized oligonucleotide primers binding at the outer DNA sequences of the gene part but in opposite orientation, e.g., with the SEQ ID NOS:2 and 3. As template T. gorgonarius DNA is used which is cleaved by restriction digestion and circularized by contacting with T4 DNA ligase. To isolate the coding region of the whole polymerase gene, another PCR is performed using primers as shown in SEQ ID NOS:4 and 5 to amplify the complete DNA polymerase gene directly from genomic DNA and introducing ends compatible with the linearized expression vector.
[0026]The gene is operably linked to appropriate control sequences for expression in either prokaryotic or eukaryotic host/vector systems. The vector preferably encodes all functions required for transformation and maintenance in a suitable host, and may encode selectable markers and/or control sequences for polymerase expression. Active recombinant thermostable polymerase can be produced by transformed host cultures either continuously or after induction of expression. Active thermostable polymerase can be recovered either from host cells or from the culture media if the protein is secreted through the cell membrane.
[0027]It is also preferable that Thermococcus gorgonarius thermostable polymerase expression is tightly controlled in E. coli during cloning and expression. Vectors useful in practicing the present invention should provide varying degrees of controlled expression of Thermococcus gorgonarius polymerase by providing some or all of the following control features: (1) promoters or sites of initiation of transcription, either directly adjacent to the start of the polymerase gene or as fusion proteins, (2) operators which could be used to turn gene expression on or off, (3) ribosome binding sites for improved translation, and (4) transcription or translation termination sites for improved stability. Appropriate vectors used in cloning and expression of Thermococcus gorgonarius polymerase include, for example, phage and plasmids. Example of phage include lambda gt11 (Promega), lambda Dash (Stratagene), lambda ZapII (Stratagene). Examples of plasmids include pBR322, pBTac2 (Boehringer Mannheim), pBluescript (Stratagene), pSP73 (Promega), pET3A (Rosenberg, A. H. et al., Gene, 56:125-135 (1987)) and pET11C (Studier, F. W. et al., Methods in Enzymology, 185:60-89 (1990)). According to the present invention the use of a plasmid has shown to be advantageously, particularly pBTac2. The Plasmid pBTac2 carrying the Thermococcus gorgonarius DNA polymerase gene is then designated pBTac2Tgo.
[0028]Standard protocols exist for transformation, phage infection and cell culture (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1982)). Of the numerous E. coli strains which can be used for plasmid transformation, the preferred strains include JM110 (ATCC 47013), LE392 pUBS 520 (Maniatis et al., supra; Brinkmann et al., Gene, 85:109-114 (1989); JM101 (ATCC No. 33876), XL1 (Stratagene), and RR1 (ATCC No. 31343), and BL21 (DE3) plysS (Studier, F. W. et al., supra). According to the present invention, the use of the E. coli strain LE392 pUBS 520 has shown to be advantageous. The E. coli strain LE392 pUBS 520 transformed with the plasmid pBTac2Tgo is then designated E. coli pBtac2Tgo (DSM No. 11328). E. coli strain XL1 Blue (Stratagene) is among the strains that can be used for lambda phage, and Y1089 can be used for lambda gt11 lysogeny. The transformed cells are preferably grown at 37° C. and expression of the cloned gene is induced with IPTG (Isopropyl-β-D-thiogalactopyranosid).
[0029]Isolation of the recombinant DNA polymerase can be performed by standard techniques. Separation and purification of the DNA polymerase from the E. coli extract can be performed by standard methods. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electric charge such as ion-exchange column chromatography, methods utilizing specific interaction such as affinity chromatography, methods utilizing a difference in hydrophobicity such as reversed-phase high performance liquid chromatography and methods utilizing a difference in isoelectric point such as isoelectric focusing electrophoresis.
[0030]One preferred method for isolating and purification of the recombinant enzyme is accomplished using the multi-stage process as follows.
[0031]The frozen cells are thawed and suspended in a suitable buffer such as buffer A (40 mM Tris-HCl, pH 7.5; 0.1 mM EDTA; 7 mM 2-mercaptoethanol) in the presence of Pefabloc SC in a final concentration of 1 mM, lysed by the addition of lysozyme (200 μg/ml) under stirring for 30 minutes at 4° C. Sodium deoxycholate is added to a final concentration of 0.05%. After an incubation for another 30 minutes, KCl is added to a final concentration of 0.75 M. The suspension is incubated at 72° C. for 15 minutes and centrifuged. The supernatant is adjusted to 25% saturation with (NH4)2SO4 and then applied to a hydrophobic interaction chromatography column such as TSK Butyl Toyopearl 650C (TosoHaas). Most of the nucleic acids and unspecific proteins are in the flow through and wash of the column while the polymerase is eluting at the end of a decreasing gradient from 30% to 0% saturation of (NH4)2SO4 in buffer A (with additional 10% glycerol). The polymerase-active fractions are pooled, dialyzed against buffer A containing 10% glycerol, adjusted to 10 mM MgCl2 and applied to a high affinity column for nucleotide-binding enzymes such as Fractogel TSK AF-Blue column (Merck). The column is washed with buffer A containing 10% glycerol and the polymerase protein is eluted with a linear gradient of 0 to 3 M NaCl in buffer A (with additional 10% glycerol). The polymerase fractions are pooled and dialyzed against the storage buffer B (20 mM Tris-HCl, pH 8.0; 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 50 mM (NH4)2SO4; 50% glycerol) and stored at -20° C.
[0032]The Thermococcus gorgonarius DNA polymerase of the present invention may be used for any purpose in which such an enzyme is necessary or desirable. For example, in recombinant DNA technology, including second-strand cDNA synthesis in cDNA cloning and DNA sequencing (see Maniatis et al., supra).
[0033]The Thermococcus gorgonarius DNA polymerase of the present invention may be modified chemically or genetically--site directed or random--to inactivate the 3'-5' exonuclease function and used for any purpose in which such a modified enzyme is desirable, e.g., DNA sequencing or DNA labeling.
[0034]In addition, the Thermococcus gorgonarius DNA polymerase of the present invention may also be used to amplify DNA, e.g., by the procedure disclosed in EP 0 200 362, EP 0 201 184 and EP 0 693 078.
[0035]The following examples are given to illustrate embodiments of the present invention as it is presently preferred to practice. It will be understood that the examples are illustrative, and that the invention is not be considered as restricted except as indicated in the appended claims.
[0036]FIG. 1: SDS polyacrylamide gel analysis of partially purified and purified recombinant DNA polymerase from T. gorgonarius.
[0037]Lane 1: 1 μl of crude extract.
[0038]Lane 2: 5 μl of polymerase fraction obtained after the first chromatography step (TSK Butyl Toyopearl 650C).
[0039]Lane 3: 5 μl of fraction obtained after second chromatography step (Fractogel Blue).
[0040]Lane 4: 10 μl of fraction obtained after second chromatography step (Fractogel Blue).
[0041]Lane 5: 10 units of DNA polymerase from Thermococcus gorgonarius.
[0042]Lane 6: Molecular weight markers.
[0043]Lane 7: 10 units of DNA polymerase from Pyrococcus woesei.
[0044]Lane 8: Molecular weight markers.
[0045]FIG. 2: In situ activity analysis of native and recombinant Thermococcus gorgonarius DNA polymerase in comparison to Klenow fragment, Pol I of E. coli and Thermus thermophilus DNA polymerase as described in Example 1. Native and recombinant Thermococcus gorgonarius DNA polymerase have the same electrophoretic mobility.
[0046]FIG. 3: DNA sequence (SEQ ID NO.:6) and the deduced amino acid sequence (SEQ ID NO.:7) of the gene encoding the DNA polymerase from Thermococcus gorgonarius.
[0047]FIG. 4: Determination of heat stability of T. gorgonarius polymerase as described in Example 5.
[0048]FIG. 5: Analysis of 3'-5' exonuclease activity as described in Example 6.
[0049]Various amounts (units are indicated in the figure) of T. gorgonarius DNA polymerase were incubated with DNA fragments in the absence (-dNTPs) and presence (+dNTPs) of desoxynucleotide triphosphates. ctrl1 and 2: Control reactions without DNA polymerase.
[0050]The 3'-5' exonuclease activity is dependent on the presence or absence of dNTPs.
[0051]FIG. 6: Comparison of various thermostable DNA polymerases (Vent exo-, 9° Nm, Taq) with respect to the incorporation of Cy5-dUTP. The reaction mixtures contained 2 mM MgCl2, 30 nM of each primer, 1 ng DNA and 200 μM deoxynucleotide. Buffer conditions were used as recommended by the supplier of the enzymes. Plasmid DNA has been used in which the β-Actin-gene of the mouse has been inserted. TTP has been partly replaced by Cy5-dUTP. The reaction mixture contained Cy5-dUTP:TTP in the following ratios: 65:35 (lane 1), 50:50 (lane 2), 35:65 (lane 3), 15:85 (lane 4). As a control the above described reaction has been performed without modified nucleosidetriphosphates (lane 5).
[0052]FIG. 7: Use of TgO-polymerase in PCR, applying different amounts of polymerase as well as different MgCl2 concentrations.
[0053]FIG. 8: Use of TgO-polymerase in PCR, applying different amounts of TgO polymerase; comparison of TgO and Pfu polymerase.
[0054]FIG. 9: Amplification of X-DNA; Comparison of TgO and Pfu polymerise.
[0055]FIG. 10: Comparison of TgO and Pfu polymerase; investigation of the influence of the KCl concentration on the PCR; 2.5 U polymerase has been used in every assay.
Example 1
[0056]Purification of a thermostable DNA polymerase from Thermococcus gorgonarius.
[0057]Thermococcus gorgonarius (DSM 8976) was grown in the medium which was prepared as follows: A mineral solution containing KCl, 325 mg/l; MgCl2 2H2O, 2.75 mg/l; MgSO4 7H2O, 3.45 mg/l; NH4Cl, 0.25 mg/l; CaCl2 2H2O, 0.15 mg/l; KH2PO4, 0.15 mg/l; NaCl, 18 g/l; NaHCO3, 1 g/l; trace elements, 4 ml/l (Balch et al., Microbiol. Rev., 43:260 (1979)), vitamins, 4 ml/l (Balch et al., supra); Rezazurin, 1 mg/l; 0.4 ml/l of a 0.2% solution of Fe(NH2)2(SO4)2 7H2O was boiled and cooled. The following components were added to the final concentrations as indicated: Peptone, 5 g/l; yeast extract, 1 g/l; Na2S.9H2O, 250 mg/l and cystein-HCl, 250 mg/l, the pH was adjusted to 6.2-6.4. The incubation temperature was 88° C. The cells were cooled to room temperature, collected by centrifugation and stored at -70° C. 6 g of cells were suspended in 12 ml of buffer A (40 mM Tris-HCl, pH 7.5; 0.1 mM EDTA; 7 mM 2-mercaptoethanol) containing 1 mM Pefabloc SCTM and disrupted by pressure at 1200 bar. KCl was added to a final concentration of 400 mM, dissolved and the solution was centrifuged at 48,200×g for 30 minutes at 4° C. The supernatant was passed through a 31 ml Heparin Sepharose Cl 6B column (Pharmacia). The column was then washed with 62 ml of buffer B (buffer A containing 10% glycerol). The column was eluted with a 310 ml linear gradient from 0 to 1.0 M NaCl in buffer B. The DNA polymerase eluted between 30 and 45 mS/cm. The fractions containing DNA polymerase activity were pooled and dialyzed twice against 600 ml buffer b, respectively, and applied to a 18 ml DEAE Sephacel column (Pharmacia). The column was washed with two column volumes of buffer b, and eluted with a 160 ml linear gradient of 0 to 0.9 M NaCl in buffer b. The polymerase activity eluted between 4 and 14 mS/cm. Fractions were pooled, dialyzed twice against buffer B (200 ml each time), and applied to a 4 ml Cellulose Phosphate P11 column (Whatman). The column was washed with 8 ml of buffer B and the activity eluted with a 40 ml linear gradient of 0 to 1 M NaCl. The active fractions which eluted between 13 and 32 mS/cm were pooled, dialyzed against buffer B containing (NH4)2SO4 to 25% saturation and applied to a 4 ml TSK Butyl Toyopearl 650C column (TosoHaas). The column was washed with 8 ml 25% (NH4)2SO4-saturated buffer B and eluted with 40 ml of a decreasing gradient of 25% to 0% (NH4)2SO4-saturated buffer b. The polymerase eluted between 74 and 31 mS/cm, the pool was dialyzed against buffer B and applied to a 4 ml Fractogel TSK AF-Orange column (Merck). The column was washed with 8 ml of buffer B and eluted with a 80 ml linear gradient of 0 to 2.0 M NaCl. The active fractions (between 76 and 104 mS/cm) were pooled and dialyzed against storage buffer C (20 mM Tris-HCl, pH 8.0; 0.1 mM EDTA; 10 mM 2-mercaptoethanol; 50 mM (NH4)2SO4; 50% glycerol) and stored at -20° C. At this step the DNA polymerase was approximately 40% pure.
[0058]The molecular weight of the isolated DNA polymerase was determined by "activity gel analysis" according to a modified version of the method described by Spanos, A. and Hubscher, U., supra. The DNA polymerase sample was separated on a SDS polyacrylamide gel containing activated calf thymus DNA. The polymerase was renaturated in the gel in 50 mM Tris-HCl, pH 8.8; 1 mM EDTA; 3 mM 2-mercaptoethanol; 50 mM KCl; 5% glycerol. Labeling of the DNA with Dig-dUTP (Boehringer Mannheim) was performed in 10 ml of the following buffer: 50 mM Tris-HCl, pH 8.8; 7 mM MgCl2; 3 mM 2-mercaptoethanol; 100 mM KCl; 12 μM dGTP; 12 μM dCTP; 12 μM dATP; 6 μM dTTP; 6 μM Dig-dUTP. The gel was first incubated under shaking at room temperature (30 minutes) and then slowly warmed up to 72° C. by temperature increments of 5° C. At each temperature interval DNA synthesis is allowed to proceed for 30 minutes, in order to detect also polymerase activity of mesophile control polymerases. Then the gel was washed and the DNA was blotted on a nylon membrane (Boehringer Mannheim), UV crosslinked. The digoxygenin-labeled DNA was detected using the protocol described in the "Boehringer Mannheim's Dig System User's Guide for Filter Hybridization". As molecular weight markers E. coli DNA polymerase I, Thermus thermophilus DNA polymerase and Klenow fragment were analyzed on the same gel. The DNA polymerase isolated from Thermococcus gorgonarius has an apparent molecular weight in the range of 92,000 to 96,000 daltons as shown in FIG. 2.
Example 2
Cloning of the T. gorgonarius DNA Polymerase
[0059]1. DNA from T gorgonarius was isolated and purified by the method described in Lawyer, F. C. et al., J. Biol. Chem., 264:6427-6437 (1989). [0060]2. The DNA was restricted with BamHI, separated on an low melting point agarose gel, denatured and blotted onto a nylon membrane. The blot was probed with a Digoxigenin labeled oligonucleotide of the sequence shown in SEQ ID NO:1. A signal could be detected and the region corresponding to the hybridization signal was cut out of the gel. The gel piece was melted and the DNA isolated by ethanol precipitation. [0061]3. The DNA fragments isolated were ligated into a plasmid vector, hybridized with SEQ ID. NO:1. The plasmid DNA from positive clones were isolated and the nucleic acid sequences of the insert determined. The DNA sequences obtained were then compared with sequences of DNA polymerase genes published in Braithwaite, D. K. and Ito, J., Nucl. Acids Res., 21:787-802 (1993). [0062]4. From the sequence of one of the cloned fragments which showed a high degree of homology to the B type DNA polymerases described in the publication of Braithwaite et al., supra, the primers SEQ ID NOS:2 and 3 were designed. These primers bind close to the ends of the cloned DNA fragment in opposite orientations to allow amplification of the flanking genomic sequences in circularized template DNA. [0063]5. With these primers "inverse PCR" was performed according of the method of Innis, M. A., supra, with the DNA from step 1 which was cleaved with EcoRI and circularized with T4 DNA ligase. With this technique two fragments were generated and the sequences determined. An open reading frame could be identified. The deduced amino acid sequence showed strong homologies to known DNA polymerases of the pol B type. [0064]6. From the sequence of the DNA fragment identified in step 5, new primers were designed, the sequences are shown in SEQ ID NOS:4 and 5, which were complementary to the start and the end of the reading frame. The primers contained additional non-complementary 5' sequences with restriction sites to introduce clonable ends into the PCR product in such an orientation that the product would be under transcriptional and translational control of the promoter. [0065]7. The PCR product was cleaved with EcoRI and PstI, purified and ligated into the vector pBTac2. This clone, expressing the DNA polymerase from Thermococcus gorgonarius was designated pBTac2Tgo.
Example 3
Expression of Recombinant T. gorgonarius DNA
[0066]The vector from example 2 was transformed into E. coli strain LE 392 pUBS 520, cultivated in a fermentor in a rich medium containing the appropriate antibiotic. Induction was performed at an optical density of 1.25 A540 with 0.5 mM IPTG. The DNA polymerase from T. gorgonarius may also be cloned and expressed by other methods.
[0067]Cells are harvested at an optical density of 5.4 A540 by centrifugation and frozen until needed or lyzed by treatment with lysozyme to produce a crude cell extract containing the T. gorgonarius DNA polymerase activity.
[0068]The crude extract containing the T. gorgonarius DNA polymerase activity is purified by the method described in Example 1, or by other purification techniques such as affinity-chromatography, ion-exchange-chromatography or hydrophobic-interaction-chromatography.
Example 4
Purification of Recombinant T. gorgonarius DNA Polymerase
[0069]E. coli (LE392 pUBS520) pBtac2Tgo (DSM No. 11328) was grown in a 101 fermentor in media containing 20 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl and 100 mg/liter ampicilline at 37° C. and induced with 0.5 mM IPTG at mid-exponential growth phase and incubated an additional 4 hours. About 45 g of cells were harvested by centrifugation and stored at -70° C.
[0070]2 g of cells were thawed and suspended at room temperature in 4 ml of buffer A (40 mM Tris-HCl, pH 7.5; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 1 mM Pefabloc SC). 1.2 mg of lysozyme were added and the cells were lyzed under stirring for 30 minutes at 4° C. 4.56 mg sodium deoxycholate were added and the suspension incubated for 10 minutes at room temperature followed by 20 minutes at 0° C. The crude extract was adjusted to 750 mM KCl, heated for 15 minutes at 72° C. and centrifuged for removal of denatured protein.
[0071]The supernatant was adjusted to 25% saturation with (NH4)2SO4 and applied to a TSK Butyl Toyopearl 650C column (1.5×10 cm; 17.7 ml bed volume) equilibrated with buffer B (buffer A containing 10% glycerol) and 30% (NH4)2SO4 saturation. The column was washed with 70 ml of buffer B and the polymerase was eluted with a 177 ml linear gradient of buffer B containing 30% to 0% (NH4)2SO4 saturation and 0 to 0.2% THESIT® (v/v).
[0072]The column fractions were assayed for DNA polymerase activity. DNA polymerase activity was measured by incorporation of digoxigenin labeled dUTP into the newly synthesized DNA and detection and quantification of the incorporated digoxigenin essentially as described below. The reaction is performed in a reaction volume of 50 μl containing 50 mM Tris-HCl, pH 8.5; 15 mM (NH4)2SO4; 7 mM MgCl2; 10 mM 2-mercaptoethanol; 100 μM of dATP, dGTP, dCTP, dTTP, respectively; 200 μg/ml BSA; 12 μg of DNAse activated DNA from calf thymus and 0.036 μM digoxigenin-dUTP and 1 or 2 Al of diluted (0.05 U to 0.01 U) DNA polymerase from T gorgonarius. The samples are incubated for 30 minutes at 72° C., the reaction is stopped by addition of 2 μl of 0.5 M EDTA, and the tubes placed on ice. After addition of 8 Al of 5 M NaCl and 150 μl of Ethanol (pre-cooled to -20° C.) the DNA is precipitated by incubation for 15 minutes on ice and pelleted by centrifugation for 10 minutes at 13,000 rpm and 4° C. The pellet is washed with 100 μl of 70% Ethanol (pre-cooled to -20° C.) and 0.2 M NaCl, centrifuged again and dried under vacuum. The pellets are dissolved in 50 μl Tris/EDTA (10 mM/0.1 mM; pH 7.5). 5 μl of the sample are spotted into a well of a nylon membrane bottomed white microwell plate (Pall Filtrationstechnik GmbH, Dreieich, FRG, product no: SM045BWP). The DNA is fixed to the membrane by baking for 10 minutes at 70° C. The DNA-loaded wells are filled with 100 μl of 0.45 μm filtrated 1% blocking solution (maleic acid, 100 mM; NaCl, 150 mM; casein, 1% (w/v); pH 7.5). All following incubation steps are done at room temperature. After incubation for 2 minutes the solution is sucked through the membrane with a suitable vacuum manifold at -0.4 bar. After repeating the washing step once the wells are filled with 100 μl of a 1:10,000-dilution of Anti-digoxigenin-AP Fab fragments (Boehringer Mannheim, FRG, No: 1 093 274) diluted in the blocking solution described above. After incubation for 2 minutes and sucking the solution through the membrane, this step is repeated once. The wells are washed twice under vacuum with 200 μl washing-buffer 1 (maleic-acid, 100 mM; NaCl, 150 mM; TWEEN® 20, 0.3% (v/v); pH 7.5). After washing for another two times under vacuum with 200 μl washing-buffer 2 (Tris-HCl, 10 mM; NaCl, 100 mM; MgCl2, 50 mM; pH 9.5) the wells are incubated for 5 minutes with 50 μl of CSPD® (Boehringer Mannheim, No: 1 655 884), diluted 1:100 in washing buffer 2 which serves as a chemiluminescent substrate for the subsequent alkaline phosphatase reaction.
[0073]The solution is sucked through the membrane and after 10 minutes incubation the RLU/s (Relative Light Unit per second) are detected in a Lummometer, e.g., MicroLumat LB 96 P (EG&G Berthold, Wilbad, FRG).
[0074]In order to correlate the relative light units to the polymerase units as defined commonly, a standard curve was prepared using a serial dilution of Taq DNA polymerase as a standard enzyme. The Taq polymerase was assayed in the buffer recommended by the supplier. The linear range of the standard curve was used to determine the relative activity of the T. gorgonarius DNA polymerase preparations.
[0075]The active fractions were pooled, dialyzed twice against 500 ml buffer B and applied to a Fractogel TSK AF-Blue column (1×10; 7.8 ml bed volume) equilibrated with buffer B. After washing with 15 ml buffer B b the column was eluted with a linear gradient of 156 ml from 0 to 3 M NaCl in buffer B supplemented with 0.05% THESIT®. The active fractions were pooled and dialyzed against the storage buffer C (20 mM Tris-HCl, pH 8.2; 10 mM 2-mercaptoethanol; 0.1 mM EDTA; 50 mM (NH4)2SO4; 50% glycerol). After adding of 0.5% of NONIDET® P40 (v/v) and 0.5% of THESIT® (v/v) the preparation was stored at -20° C.
Characterization of the Recombinant DNA Polymerase from Thermococcus gorgonarius
[0076]Recombinant and native T. gorgonarius DNA polymerase had the same apparent molecular weight when electrophoresed in 8-25% SDS-PAGE gradient gels. Recombinant T. gorgonarius polymerase maintains the heat stability of the native enzyme. Recombinant T. gorgonarius polymerase has the same 3'-5' exonuclease activity as native T. gorgonarius polymerase, which is also sensitive to inhibition by an excess of dNTPs.
Example 5
Thermostability of T. gorgonarius DNA Polymerase
[0077]The thermostability of the DNA polymerase from T. gorgonarius purified as described in Example 1 was determined as follows: 5 units purified T. gorgonarius polymerase were incubated at 95° C. in 100 gl of the following buffer: 50 mM Tris-HCl, pH 8.8 (at 25° C.); 15 mM (NH4)2SO4; 7 mM MgCl2; 10 mM 2-mercaptoethanol; 200 μM each of dATP, dGTP, dCTP and dTTP; 0.1% NONIDET® P40, 0.1% THESIT®; 25 μg DNAse treated calf thymus DNA. 15 μl samples were taken at 0, 5, 10, 15, 30, 45, 60 and 120 minutes. The remaining polymerase activity was measured as described in Example 4 by determining incorporation of labeled 3H-TTP into DNA in a 50 μl volume of the incubation mixture described above containing in addition 150 nCi of 3H-TTP. After incubation at 72° C. for 30 minutes, the reactions were stopped by addition of 300 μl 10% TCA, and after 10 minutes at 0° C. the mixtures were applied onto 3mM filters (Whatman). The filters were washed three times with approximately 10 ml 5% TCA each time, dried for 10 minutes at 75° C. and the DNA-bound radioactivity of each filter was measured in 5 ml scintillation liquid in a scintillation vial in LKB rack beta 1217/1218 (Pharmacia).
[0078]As shown in FIG. 4, the T. gorgonarius DNA polymerase retained almost 90% of its initial activity after incubation for 120 minutes at 95° C., Pwo polymerase has a similar stability, while Taq DNA polymerise has a remaining activity of approximately 16% only.
Example 6
Determination of 3'-5' Proofreading Activity
[0079]A series of units of T. gorgonarius DNA polymerase (see FIG. 5) were incubated for 4 hours at 72° C. with 1 μg DNA molecular weight marker VI (Boehringer Mannheim) in the presence and absence of dNTPs, 1 mM each, in 50 μl of the following incubation buffer: 50 mM Tris-HCl, pH 7.8; 10 mM MgCl2; 7 mM 2-mercaptoethanol with Paraffin overlay. After addition of 10 μl stop solution the DNA fragments were separated on a 1% agarose gel. In the absence of dNTPs, a smear of DNA fragments or no DNA could be detected while in presence of dNTPs the DNA fragments remained undegraded.
Example 7
Fidelity of T. gorgonarius DNA Polymerase in the PCR Process
[0080]The fidelity of T. gorgonarius DNA polymerase in the PCR process was determined in an assay based on the amplification, circularization and transformation of the pUC19 derivate pUCIQ17, containing a functional lac Iq allele (Frey, B. and Suppmann, B., Biochemica, 2:34-35 (1995)). PCR-derived mutations in lac I are resulting in a derepression of the expression of lac Zα and subsequent formation of a functional β-galactosidase enzyme which can be easily detected on X-Gal indicator plates. The error rates determined with this lac I-based PCR fidelity assay were in the range of 3.4 to 2.210-6.
[0081]The plasmid pUCIQ17 was linearized by digestion with DraII to serve as a substrate for PCR amplification with DNA polymerase of T. gorgonarius. Both of the primers used have ClaI sites at their 5 prime ends:
TABLE-US-00003 SEQ ID NO: 8: Primer 1: 5'-AGCTTATCGATGGCACTTTTCGGGGAAATGTGCG-3' SEQ ID NO: 9: Primer 2: 5'-AGCTTATCGATAAGCGGATGCCGGGAGCAGACAAGC-3'
[0082]The length of the resulting PCR product is 3493 pb.
[0083]The PCR was performed in a final volume of 50 μl in the presence of 1.5 mM MgCl2, 50 mM Tris-HCl, pH 8.5 (25° C.), 12.5 mM (NH4)2SO4, 35 mM KCl, 200 μM dNTPs and 2.5 units of T. gorgonarius DNA polymerase. Conditions of the amplification reaction using T. gorgonarius DNA polymerase are
[0084]The cycle conditions were as follows:
1 × denaturation of template for 2 min . at 95 ° C . 8 × [ denaturation at 95 ° C . for 10 sec . annealing at 57 ° C . for 30 sec . elongation at 72 ° C . for 4 min . 16 × [ denaturation at 95 ° C . for 10 sec . annealing at 57 ° C . for 30 sec . elongation at 72 ° C . for 4 min . + cycle elongation 20 sec . for each cycle ##EQU00001##
[0085]After PCR, the PCR products were PEG-precipitated (Barnes, W. M., Gene, 112:229 (1992)) the DNA restricted with ClaI and purified by agarose gel electrophoresis. The isolated DNA was ligated using the Rapid DNA Ligation Kit (Boehringer Mannheim GmbH) and the ligation products transformed in E. coli DH5a, plated on TN Amp X-Gal plates. The α-complementing E. coli strain DH5a transformed with the resulting plasmid pUCIQ17 (3632 bp), shows white (lacI.sup.+) colonies on TN plates (1.5% Bacto Tryptone, 1% NaCl, 1.5% Agar) containing ampicillin (100 μg/ml) and X-Gal (0.004% w/v). Mutations result in blue colonies.
[0086]After incubation overnight at 37° C., blue and white colonies were counted. The error rate (f) per by was calculated with a rearranged equation as published by Keohavong and Thilly (Keohavong, P. and Thilly, W., PNAS USA, 86:9253 (1989)):
f=-ln F/d×bbp
where F is the fraction of white colonies:
F=white (lacI+)colonies/total colony number;
d is the number of DNA duplications:
2d=output DNA/input DNA;
and b is the effective target size of the (1080 bp) lac I gene, which is 349 by according to Provost et al. (Provost et al., Mut. Res., 288:133 (1993)).
Example 8
Fidelity Assay
[0087]Determination of the misincorporation rates of DNA polymerases from Pyrococcus furiosus and Thermococcus gorgonarius under PCR conditions.
[0088]Error rates of many DNA polymerases are published. For example for the DNA polymerase of Pyrococcus furiosus various error rates were measured (Lit. 1-5). They may vary with the conditions used, e.g., nucleotide triphosphate concentrations, enzyme preparation, buffer conditions and of course with the method used, the determination of the number of duplications and the way to calculate the misincorporation rate.
[0089]Therefore, the DNA polymerases Pfu (Stratagene) and Tgo (Boehringer Mannheim GmbH) were analyzed in parallel in the same system (Protocol: Frey, B. and Suppman, B. Boehringer Mannheim Biochemica Information, Nr. 96-1995, 21-23).
TABLE-US-00004 TABLE 1 Fidelity of Pfu and Tgo DNA polymerases in PCR fidelity assay DNA Plaques scored Mutation Error Polymerase Total Mutant frequency Error rate (a) rate (b) Pfu 1. sample 3082 76 2.47 1.56 × 10-5 8.2 × 10-6 2. sample 2693 68 2.52 1.6 × 10-5 8.4 × 10-6 Tgo 1. sample 1904 12 0.63 3.5 × 10-6 1.8 × 10-6 2. 2003 20 1 5.6 × 10-6 2.9 × 10-6
[0090](a) Error rate calculated according to the equation used by Stratagene (Lundberg, K. S. et al., Gene, 108:1-6 (1991)).
[0090]ER=mf/bp×d [0091]ER=error rate [0092]mf=mutation frequency in % minus background frequency of 0.0017% [0093]bp is the number of detectable sites in the lac I gene sequence (182) [0094]d is the number of duplications. In this particular experiment the number of duplications was determined/estimated for Pfu to be 8.64 and for Tgo to be 9.64 [0095](b) Error rate calculated per by with a rearranged equation published by Keohavong, P. and Thilly, W., PNAS USA, 86:9253 (1989).
[0095]ER=-ln F/d×bbp [0096]F=fraction of white colonies (white colonies/total number of colonies) [0097]d=the number of duplications. 2d=output DNA/input DNA [0098]b is the effective target size of the (1080 bp) lac I gene, which is 349 by according to Provost, G. S. et al., Mut. Res., 288:133 (1993)
[0099]These data show that the mutation frequency of Tgo DNA polymerase is lower than that of Pfu, and the fidelity (calculated in errors per base pair) is higher no matter which way of calculation was used.
References describing error rates for Pfu: [0100]1. Lundberg, K. S. et al., Gene, 108:1-6 (1991) (1.6×10-6 errors/base) [0101]2. Flaman, J. M. et al., NAR, 22:3259-3260 (1994). (2×10-6 errors/base) For Tli (Vent) Polymerase: (Variations in error rate depending on assay) [0102]3. Cariello, N. F. et al., NAR, 19:4193-4198 (1991). (2.4×10-5 errors/base) [0103]4. Ling, L. L. et al., PCR Methods Appl., 1:63-69 (1991). (4.5×10-5 errors/base) [0104]5. Matilla, P. et al., NAR, 19:4967-4973 (1991). (5.7×10-5 errors/base)
Sequence CWU
9126DNAArtificialprobe 1atgathytng ayacngayta yathac 26224DNAArtificialamplification primer 2ggcctacgag aggaacgaac tggc 24318DNAArtificialamplification primer 3ggcgtagatg tagggctc 18442DNAArtificialamplification primer 4gagctggtcg aattcatgat cctggacgct gactacatca cc 42533DNAArtificialamplification primer 5agcctgcagt catgtcttag gttttagcca cgc 3362322DNAThermococcus gorgonariusCDS(1)..(2322) 6atg atc ctc gat aca gac tac ata act gag gat gga aag ccc gtc atc 48Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile1 5 10 15agg atc ttc aag aag gag aac ggc gag ttc acc ata gac tac gac aga 96Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Asp Tyr Asp Arg 20 25 30aac ttt gag cca tac atc tac gcg ctc ttg aag gac gac tct ccg att 144Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Pro Ile 35 40 45gag gac gtc aag aag ata act gcc gag agg cac ggc act acc gtt agg 192Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Thr Val Arg 50 55 60gtt gtc agg gcc gag aaa gtg aag aag aag ttc cta ggc agg ccg ata 240Val Val Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile65 70 75 80gag gtc tgg aag ctc tac ttc act cac ccc cag gac gtt ccc gca atc 288Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95agg gac aag ata aag gag cat cct gcc gtt gtg gac atc tac gag tac 336Arg Asp Lys Ile Lys Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110gac atc ccc ttc gcg aag cgc tac ctc ata gac aaa ggc tta atc ccg 384Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125atg gag ggc gac gag gaa ctt aag atg ctc gcc ttc gac atc gag acg 432Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140ctc tat cac gag ggc gag gag ttc gcc gaa ggg cct atc ctg atg ata 480Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile145 150 155 160agc tac gcc gac gag gaa ggg gcg cgc gtt att acc tgg aag aat atc 528Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175gac ctt ccc tat gtc gac gtc gtt tcc acc gag aag gag atg ata aag 576Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190cgc ttc ctc aag gtc gtc aag gaa aag gat ccc gac gtc ctc ata atc 624Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Ile 195 200 205tac aac ggc gac aac ttc gac ttc gcc tac ctc aag aag cgc tcc gag 672Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220aag ctc gga gtc aag ttc atc ctc gga agg gaa ggg agc gaa ccg aaa 720Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Glu Gly Ser Glu Pro Lys225 230 235 240atc cag cgc atg ggc gat cgc ttt gcg gtg gag gtc aag gga agg att 768Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255cac ttc gac ctc tac ccc gtc att agg aga acg att aac ctc ccc act 816His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270tac acc ctt gag gca gta tat gaa gcc atc ttt gga cag ccg aag gag 864Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu 275 280 285aag gtc tac gct gag gag ata gcg cag gcc tgg gaa acg ggc gag gga 912Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Thr Gly Glu Gly 290 295 300tta gaa agg gtg gcc cgc tac tcg atg gag gac gcg aag gta acc tat 960Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr305 310 315 320gaa ctc gga aaa gag ttc ttc cct atg gaa gcc cag ctc tcg cgc ctc 1008Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335gta ggc cag agc ctc tgg gat gta tct cgc tcg agt acc gga aac ctc 1056Val Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350gtc gag tgg ttt ttg ctg agg aag gcc tac gag agg aat gaa ctt gca 1104Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365cca aac aag ccg gac gag agg gag ctg gca aga aga agg gag agc tac 1152Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Glu Ser Tyr 370 375 380gcg ggt gga tac gtc aag gag ccc gaa agg gga ctg tgg gag aac atc 1200Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile385 390 395 400gtg tat ctg gac ttc cgc tcc ctg tat cct tcg ata ata atc acc cat 1248Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415aac gtc tcc cct gat aca ctc aac agg gag ggt tgt gag gag tac gac 1296Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Glu Glu Tyr Asp 420 425 430gtg gct cct cag gta ggc cat aag ttc tgc aag gac ttc ccc ggc ttc 1344Val Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445atc cca agc ctc ctc gga gac ctc ttg gag gag aga cag aag gta aag 1392Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Val Lys 450 455 460aag aag atg aag gcc act ata gac cca atc gag aag aaa ctc ctc gat 1440Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Lys Lys Leu Leu Asp465 470 475 480tac agg caa cga gca atc aaa atc ctt gct aat agc ttc tac ggt tac 1488Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495tac ggc tat aca aag gcc cgc tgg tac tac aag gag tgc gcc gag agc 1536Tyr Gly Tyr Thr Lys Ala Arg Trp Tyr Tyr Lys Glu Cys Ala Glu Ser 500 505 510gtt acc ggt tgg ggc agg gag tac atc gag acc acg ata agg gaa ata 1584Val Thr Gly Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile 515 520 525gag gag aaa ttt ggc ttt aaa gtc ctc tac gcg gac aca gat gga ttt 1632Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540ttc gca aca ata cct gga gcg gac gcc gaa acc gtc aaa aag aag gca 1680Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala545 550 555 560aag gag ttc ctg gac tac atc aac gcc aaa ctg ccc ggc ctg ctc gaa 1728Lys Glu Phe Leu Asp Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu 565 570 575ctc gaa tac gag ggc ttc tac aag cgc ggc ttc ttc gtg acg aag aag 1776Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590aag tac gcg gtt ata gac gag gag gac aag ata acg acg cgc ggg ctt 1824Lys Tyr Ala Val Ile Asp Glu Glu Asp Lys Ile Thr Thr Arg Gly Leu 595 600 605gaa ata gtt agg cgt gac tgg agc gag ata gcg aag gag acg cag gcg 1872Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620agg gtt ctt gag gcg ata cta aag cac ggt gac gtt gaa gaa gcg gta 1920Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val625 630 635 640agg att gtc aaa gag gtt acg gag aag ctg agc aag tac gag gtt cca 1968Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655ccg gag aag ctg gtc atc tac gag cag ata acc cgc gac ctg aag gac 2016Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asp Leu Lys Asp 660 665 670tac aag gcc acc ggg ccg cat gtg gct gtt gca aaa cgc ctc gcc gca 2064Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685agg ggg ata aaa atc cgg ccc gga acg gtc ata agc tac atc gtg ctc 2112Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700aaa ggc tcg gga agg att ggg gac agg gct ata ccc ttt gac gaa ttt 2160Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe705 710 715 720gac ccg gca aag cac aag tac gat gca gaa tac tac atc gag aac cag 2208Asp Pro Ala Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735gtt ctt cca gct gtg gag agg att ctg agg gcc ttt ggt tac cgt aaa 2256Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750gaa gat tta agg tat cag aaa acg cgg cag gtt ggc ttg ggg gcg tgg 2304Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp 755 760 765cta aaa cct aag aca tga 2322Leu Lys Pro Lys Thr 7707773PRTThermococcus gorgonarius 7Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile1 5 10 15Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Thr Ile Asp Tyr Asp Arg 20 25 30Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Pro Ile 35 40 45Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Thr Val Arg 50 55 60Val Val Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile65 70 75 80Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95Arg Asp Lys Ile Lys Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile145 150 155 160Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Ile 195 200 205Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Glu Gly Ser Glu Pro Lys225 230 235 240Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu 275 280 285Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Thr Gly Glu Gly 290 295 300Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr305 310 315 320Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335Val Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Glu Ser Tyr 370 375 380Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile385 390 395 400Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Glu Glu Tyr Asp 420 425 430Val Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Val Lys 450 455 460Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Lys Lys Leu Leu Asp465 470 475 480Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr 485 490 495Tyr Gly Tyr Thr Lys Ala Arg Trp Tyr Tyr Lys Glu Cys Ala Glu Ser 500 505 510Val Thr Gly Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile 515 520 525Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala545 550 555 560Lys Glu Phe Leu Asp Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu 565 570 575Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590Lys Tyr Ala Val Ile Asp Glu Glu Asp Lys Ile Thr Thr Arg Gly Leu 595 600 605Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val625 630 635 640Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asp Leu Lys Asp 660 665 670Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe705 710 715 720Asp Pro Ala Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp 755 760 765Leu Lys Pro Lys Thr 770834DNAArtificialamplification primer 8agcttatcga tggcactttt cggggaaatg tgcg 34936DNAArtificialamplification primer 9agcttatcga taagcggatg ccgggagcag acaagc 36
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text:
The funding comes from the School Development Authority's (SDA) Regular Operating District (ROD) grant program, which assists non-Abbott school districts with major capital improvements that address health and safety issues, student overcrowding and other critical needs.
"The roof is over ten years old and it was starting to deteriorate in areas and repairs were needed," said Superintendent Henry Bermann. "We applied for the grant in cooperation with Garrison Architects."
Bermann said the roof work was completed over the summer and finished more than a month ago.
"The roof was leaky in certain spots like the media center and some of the hallways and classrooms," said Bermann. "The leaks were in more places then we wanted but it wasn't to the point where we needed buckets or umbrellas."
The push for a new roof stemmed from the district wanting to take a proactive approach to avoid moisture problems, he said.
"I've been told after the past couple rains there is no more spotted tiles," said Bermann.
Along with a new roof, the district replaced 18 exhaust fans.
"They were over 30 years old," he said.
The Pittsgrove School District will receive a $542,986 state grant to help finance the roof replacement project at the Arthur P. Schalick High School.
With the ROD grant, the state will be picking up 52 percent of the total cost of the roof replacement, estimated at $987,500.
"Aside from providing a much-needed repair, this grant will ensure that the burden of improving the high school is not shouldered entirely by property taxpayers," said Assemblyman John J. Burzichelli. "Replacing a roof is one of the costliest capital projects a district can take on, and having this help from Trenton will go a long way."
Bermann said the remaining cost, roughly $444,000, to complete the roof was bonded over a five-year period — something he said was much more manageable for the district than borrowing nearly $1 million for the entire cost of the roof.
"Borrowing for roughly $100,000 a year over five years for capital improvements is much more manageable for us," Bermann said.
Since May 2010, the SDA has executed 704 ROD grants. The state has provided nearly $198 million in grants to local districts, leveraging projects estimated at a total of $434.7 million.
"This grant will ensure that Schalick High School remains a safe and secure learning environment for our children," said Senate President Steve Sweeney. "I thank the SDA for partnering with Pittsgrove to make this vital improvement possible."
Funding for the program was made available through the sale of $500 million in bonds by the New Jersey Economic Development Authority (EDA).
"While the students and staff at Schalick High School will be the direct beneficiaries of this work, property taxpayers will also be long-term winners," said Assemblywoman Celeste Riley. "A new, modern roof also helps lower heating and cooling costs, and will allow the district to save money over time."


text:
2 Salem cops who died on duty decades ago nominated for memorial
SALEM -Two Salem police officers who lost their lives while serving citizens in the early 1900s are being nominated for inclusion on The National Law Enforcement Officers Memorial in Washington, D.C.
"Obviously they gave the ultimate sacrifice, dying in the line of duty," Chief J.T. Panezott said.
Officer Charles Miller was shot and killed by a burglary suspect shortly after arresting him at the Pennsylvania train depot on April 8, 1908. Patrolman Edward Piller died April 25, 1936 in an accident while testing out the department's recently repaired motorcycle.
Panezott said he was contacted earlier this year by a researcher from the National Law Enforcement Memorial Fund who's also a police officer in Pennsylvania and asked if the department had any officers killed in the line of duty.
The researcher already knew about Miller, but then the chief asked about Piller and the researcher did research on both men with the printed material the department had on hand and by visiting the Salem Historical Society for assistance.
According to the website at www.nleomf.org, the National Law Enforcement Officers Memorial Fund was founded in 1984 to honor and remember the service and sacrifice of law enforcement officers in the United States. The memorial, which lists the names of more than 19,000 officers killed in the line of duty dating back to the first known officer killed in 1791, was dedicated on Oct. 15, 1991.
Panezott said they want to see that Miller and Piller get honored for their sacrifice. The stories of both men have become part of the records of the late Salem historian Dale Shaffer, who wrote about their deaths. Some of that background information will be submitted with the applications.
The website which explained the nomination and approval process noted that "the term "killed in the line of duty" means a law enforcement officer has died as a direct and proximate result of a personal injury sustained in the line of duty. This includes victim law enforcement officers who, while in an off-duty capacity, act in response to a law violation. It also includes victim law enforcement officers who, while in an off-duty capacity, are en route to or from a specific emergency or responding to a particular request for assistance; or the officer is, as required or authorized by law or condition of employment, driving his or her employer's vehicle to or from work; or when the officer is, as required by law or condition of employment, driving his or her own personal vehicle at work and is killed while en route to or from work."
Miller was the first Salem officer killed in the line of duty and had been on the job just three years. Police had been called about some burglaries at homes on South Lincoln Avenue and when a ticket agent called about a suspicious person at the train depot, they gave him a description of the alleged burglary and told him to be on the lookout.
The story by Dale Shaffer said the ticket agent spotted a man fitting the description given by police and called them. Miller arrived and found the burglar on a passenger train car and took him into custody. A witness said that after they stepped down from the train onto the platform, the prisoner "suddenly switched his hand to his hip pocket, drew his gun, turned, pressed it against Miller, and fired two shots."
Officer Miller fell dead on the spot and is buried in Hope Cemetery, where his grave marker recognizes his death in the line of duty as a Salem police officer.
Piller, who had been an officer about six years, died at the Central Clinic of a fractured skull and concussion after being injured in the motorcycle accident several hours earlier. The mayor at the time, George Harroff, had also been on the motorcycle and was injured, but survived.
Piller was a native of Washingtonville and according to an account by Shaffer, he was putting the repaired motorcycle through its paces after it had been out of service about six months. Chief of Police Ralph Stoffer rode the department's new motorcycle with them when Piller and Harroff decided to go for a short ride and was in the lead when he looked back and saw the bike go into the air and throw the riders off head-first.
Piller is also buried at Hope Cemetery.
Approval of the nominations may not come until early 2014, with new names added to the memorial in the spring.


text: Home > Entertainment > Bollywood The Attacks Of 26/11: Pros and Cons Anu ChanchalMarch 1, 2013 facebook twitter reddit gplus SKIP AD Image 1 of 7 Related Galleries Next Gallery Starts in 12 Secs X SKIP WAITING AUTOPLAY ON OFF Prev Next Bollywood A-Listers Flock To Karan Johar's Fashion Show Vanity Insanity! Bollywood Actors Snapped While Taking Selfies 13 Years Later These Quotes Won't Make Us Forget The Tragedy That Was 9/11 FIRST LOOK: Rahul Khanna, Monica Dogra In Fireflies Fawad Khan: I Am Not A Very Talkative Person Arjun, Deepika, Sonam, Kalki Go Shop Shop Shop 68628 Prev Next Based on the ghastly 2008 Mumbai terror attacks, RGV's The Attacks Of 26/11 has already created a storm in cine-circles - quite obviously. Though the high-voltage drama has more robust points attached, we certainly can't overlook the weedy ones. Let's check out both the sides... PROS:PLOT - Ram Gopal Varma's crime-thriller will surely see packed-cinema halls, at least in the opening week, courtesy the film's storyline that is rooted in the appalling 2008 Mumbai terror attacks. That whether the script is watertight and moving enough to tug at our heartstrings, will be reported only after the first few days of its release. CAST - With the first-rate emoter like Nana Patekar, the film promises to be a great experience. The brilliant actor plays the Joint Commissioner of Police (Crime), and has been able to strike a chord with the cinema-loyalists – thanks to few glimpses of his super intense shots in the promos. As for the face of terror from the brutal attacks, a budding actor, Sanjeev Jaiswal has slipped into the prime accused terrorist Ajmal Kasab's role. Endowed performers Atul Kulkarni and Ganesh Yadav too are in pivotal parts. SUPER-SUCCESSFUL TRAILER -The Attacks Of 26/11 easily piqued the nation's curiosity, right from its announcement, for obvious reasons. And so, when RGV launched the promo video of the first seven minutes of his film online, the eager beavers couldn't hold themselves back from sneaking a peek into it. As a result, those few minutes of the cinematic version of the dreadful event, soon crossed a million views on YouTube! Clearly, the expectations are sky-high from the full screen-interpretation now. BOX OFFICE CLASH -Competing with Ramu's crime-saga at the ticket-windows, is John-Chitrangada-Prachi-starrer romantic-drama I, Me Aur Main. The rivalry is certainly not stiff, as the latter (given the glossy trailers) appears to be no different from the typical confused relationship tales that involve good-looking people but fail to boast of a heart and a soul. Under this impression, The Attacks Of 26/11 has the upper-hand, at the outset at least. CONS:RGV'S FAILING GRIP ON DIRECTION -In his attempt to recreate one of the most disastrous events in Indian History, RGV has evidently shouldered a huge responsibility. But a close look at his recent cinema-graphy, and we are afraid to say that the director hasn't been in top form of late. Not A Love Story, Bhoot Returns and Department were big disasters and disappointments, despite their big-time promotions. On that note, it is for all to now witness whether Ram Gopal Varma has completely lost his edge or he manages to earn his spurs in the battle this time around. If the film succeeds at charming the BO cash registers, it will indeed be a major comeback for the maverick filmmaker. SOUNDRACK -The crime-drama's tracks have failed to stay with the listeners for long. Except for Maula Maula, rest of the numbers have easily fallen short of gripping cine-buffs' attention. Going by the fact that a film's background score and songs play a good part in engaging the viewers, The Attacks Of 26/11 has already lost a few points. < 1 2 3 4 5 > Follow @indiatimes facebook twitter reddit gplus Comment Here Based on the ghastly 2008 Mumbai terror attacks, RGV's The Attacks Of 26/11 has already created a storm in cine-circles - quite obviously. Though the high-voltage drama has more robust points attached, we certainly can't overlook the weedy ones. Let's check out both the sides... ADVERTISEMENT


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Global Justice
Waste is much more than a technical problem: it is part of a larger web of health, equity, race, power, gender, poverty, and governance issues.
Because of this, we only achieve true solutions when our work supports systems of social and economic justice and ecological sustainability. Without an understanding of this broader context, policy-makers can fall into the trap of seeing "waste" primarily as a technical problem awaiting the next magic solution. Then an incinerator or "sanitary landfill" can be perceived as a solution, leaving the fundamental model of our economies, which depend on a high level of wasting of both the planet and its communities, unquestioned.
The good news is that severe threats to human health and livelihoods often inspire strong grassroots action and motivate people to come together in defense of their communities. And although the wealth and lobbying power of the waste industry is great, the victories of GAIA's members and allies are many.
GAIA believes that we will never win unless we see our specific struggle as inseparable from that of the broader movements toward social and economic justice and ecological sustainability. We are not fighting incinerators just to stop incinerators, but because we see stopping incinerators as a strategic leverage point to advance a deeper transformation of society. We absolutely cannot stop toxic pollution without simultaneously promoting environmental and social justice, because pollution depends on and perpetuates injustice. Because of this, supporting the principles of environmental justice is a key component in our work for zero waste.
As one GAIA member explained: We need to turn NIMBY (not in my backyard) to NOPE (not on planet Earth).