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Radius: ... miles
Sun 10th Nov at 8:00pm
Josh Widdicombe
@ Tobacco Factory, Bristol
Also appearing: Suzi Ruffell
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Never miss another 'Josh Widdicombe' show
Incidentally...: Josh Widdicombe, Suzi Ruffell
Josh Widdicombe returns to the road this autumn touring his brand new show Incidently…
The last twelve months have been incredible for Josh Widdicombe, with the hit Channel 4 show The Last Leg being commissioned for two series after its success during the Paralympics Games. Widdicombe is fast becoming a TV regular with appearances on Channel 4's 8 Out Of 10 Cats and BBC1's Live At The Apollo and Mock The Week.
Josh Widdicombe
One of the best up-and-coming comedians on the UK circuit, Josh Widdicombe is in constant demand at clubs and theatres across Britain. He is also fast establishing himself on the live circuit, regularly supporting huge comic talent such as Alan Carr, Kevin Bridges and Michael McIntyre. Star of BBC2's 'The Rob Brydon Show', Channel 4's 'Stand Up For The Week' and '8 Out Of 10 Cats', Josh was also nominated for Fosters Edinburgh Comedy Awards Best Newcomer 2011 and Chortle Best Breakthrough Act in 2011. He was also FHM Stand-Up Hero 2010.
'Guardian sports reporter turned ace observationalist Josh Widdicombe has the ability to find the sparkle in the mundane that puts him in line to be the next Sean Lock' (The Guardian)
'This is one of the most individual, accomplished, hilarious and unforgettable hours I have had this year' (The Scotsman)
'His talent for comedy is priceless' (Evening Standard)
'Nicely crafted observational comedy' (The Independent).
Josh Widdicombe tour dates
Suzi Ruffell
An innovative and immensely likeable comic who was a Laughing Boy New Act Of The Year.
Performing all over the country Suzi has quickly established herself supporting Alan Carr, Josh Widdicombe and Kevin Bridges on tour.
Ruffell's first television appearance on BBC3's Ed Comedy Fest Live and is soon to be performing on Comedy Centrals 'Live From The Comedy Store'.
She also recently recorded a two hour XFM Comedy Special as one of their featured comedy rising stars.
'Suzi's great. A lovely stage persona, hugely likable and definitely one to watch!' (Time Out)
'A real stand-up gem' (The Guardian).
Suzi Ruffell tour dates
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Venue Facts
Venue: Tobacco Factory
Website: tobaccofactorytheatres.com
Phone: 0117 902 0344
→ Parson Street 14 min walk
→ Bedminster 17 min walk


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Aerospace Professional Services, LLC filed as a Domestic Limited Liability Company (LLC) in the State of Texas on Monday, January 18, 2010 and is approximately five years old, according to public records filed with Texas Secretary of State. The filing is currently active as of the last data refresh which occured on Saturday, July 4, 2015.
Timothy Gutierrez serves as the Managing Member and has interests in other corporate entities including All American Target Concepts, LLC located in Haslet, TX .
The registered agent for the company is National Registered Agents, Inc.. Also known as a statutory or resident agent, the registered agent is responsible for receiving legal notifications regarding court summons, lawsuits, and other legal actions involving the corporate entity.
Key roles for Aerospace Professional Services, LLC
Timothy J. Gutierrez
Managing Member
|Filing Type:
|Domestic Limited Liability Company (LLC)
|State ID:
|Date Filed:
|Monday, January 18, 2010
|Registered Agent
|National Registered Agents, Inc.
Comprehensive Insight Plus Report
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|The D&B Comprehensive Insight Plus Report for Aerospace Professional Services, LLC combines D&B's proprietary statistical scoring with business, payment and financial information all in one report.
US Company Profile Report
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|The D&B U.S. Company Profile contains basic information such as address and telephone number, executive names and titles, line of business and SIC codes, D-U-N-S Numbers, organization status, and year and location of incorporation for Aerospace Professional Services, LLC.

subset: commoncrawl


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Editing and Maintaining Parcels Using ArcGIS
- Format: Instructor-Led
- Duration: 2 days (16 hours)
- Price: $1,070 USD
- ArcGIS Version: 10.1, 10.2, 10.3
- Authored by Esri
This course teaches techniques to efficiently store, edit, and ensure the accuracy of land-records data. Using the ArcGIS parcel fabric and Local Government Information Model, you will learn recommended workflows to perform many common parcel-editing tasks.
Note: Course concepts apply to ArcGIS 10.1 Service Pack 1, ArcGIS 10.2, and ArcGIS 10.3. Esri will provide either ArcGIS 10.2 or 10.3 for use during course exercises. The software version used in class is shown on the schedule.
GIS technicians, parcel editors, tax mapping professionals, and others who maintain or manage land-records data
- Familiarity with land records terminology and practices is required.
- Completion of ArcGIS 2: Essential Workflows or equivalent knowledge is required.
- Completion of Building Geodatabases or equivalent knowledge is recommended.
New Catalog Search
Upcoming Classes
- Jul 27 - Jul 28, 2015
- Jul 29 - Jul 30, 2015
- Aug 26 - Aug 27, 2015
- Sep 30 - Oct 01, 2015
- Nov 23 - Nov 24, 2015
Speak with a Specialist
We can answer questions and help you schedule a class that meets your needs. Call 800-447-9778, ext. 5757 or email us.
I thought the course was well put together and gave me a great amount of knowledge that I will be using in my everyday work.
Kelly Felton, Wisconsin


text:
A group of families striving to provide an exceptional learning environment for their kids, based on their kids' interests and developmental abilities, and a healthy, fruity lifestyle.
Members: 67
Latest Activity: Sep 28, 2014
Started by Mera/Feather Story. Last reply by Mary Mar 25, 2014. 1 Reply 0 Likes
If you guys have unschooling(or related blogs) please post a link below.Currently my blog doesn't have much about unschooling on it but it will soon.…Continue
Started by Mary. Last reply by Mary Sep 5, 2013. 2 Replies 0 Likes
I think my daughter is still wanting to go to school. She is 8 and I think she misses mostly the social part, though we do get out a lot and see lots of people, there are not a whole lot of girls in…Continue
Started by Windlord. Last reply by Aime May 31, 2013. 2 Replies 1 Like
For those interested, I recently found this in my library:Home Education Magazinehttp://homeedmag.com/Continue
Started by Sophia S. Last reply by Andrea Grace May 30, 2013. 6 Replies 0 Likes
I just started unschooling/homeschooling back in march and am in my 3rd year of high school. I'm so worried about college. I want to be able to learn and do whatever I want but I keep thinking about…Continue
Hi guys, wanted to share my homeschool support blog - www.homeschoolhottub.com
Katt, thanks for the ted talks link, that was great!
This is the best time to be an unschooler/homeschooler. At least in this hemisphere, spring/summer is in the air, we can be outside as much as we want, we're gardening, hiking, looks like this coming Tuesday will be our first beach weather day, yeah? Not sitting inside a school for 7 hours, that's what we're talking about.......
Hi.. i teach in the public school system,
but i am so for homeschool, unschooling, and this.. :-)
a must watch - School in the clouds..
talks about how education as we know it is obsolete,and has been for a loonngg time. and a new approach.. absolutely amazing...his experiments in India with the children of the slums..
Sunshine, I like the feelings poster but what's with the last one "beauty"? It doesn't seem to belong with the other feelings.
As an homeschooler/unschooler I am constantly questioning myself - Am I doing this right; am I screwing my kids up; am I doing enough; how long can I keep this up; etc etc. I guess it's a good thing to question the status quo and keep trying to improve - maybe that's what education leaders should be doing!
That's beautiful Mary. Good for you letting your kids be themselves. Them knowing you appreciate their hard work will motivate them even more to excel.
This 7-year-old unschooler taught himself piano one day. He sat for four hours (which is really unlike hime!) and plowed through the Thompson's 1st level. Hasn't played since but he's taking guitar lessons now.
I would never send my kids to Indoctrination camp. Way better to spend time with them and learn from each other, while being very close and connected as well.
"The more you can let go of old habits and expectations, the more more enjoyable it becomes."
This is the TRUTH!! Thanks for that insight. = )
I have a new friend I hang out with these days, and she is very tightly wound with her parenting. She spends too much time yelling at her son for playing with dirt, running too far, climbing up on something, etc. She adores him and means well but sorta wears the Strict Mom title as a badge of honor. Meanwhile my daughter plays alongside him and gets into the same stuff he does, but I don't stop her. My friend spends much more time chasing after him, chastising him, scolding him. Her tone has a sharp edge that caries in public and sounds quite harsh. I don't do any of that and use much less energy and carry a softer tone. Now, maybe she just thinks I'm irresponsible or careless, but it's not true. I'm always alert; I just choose to trust Ana and show her my trust by letting her explore. I hope this might rub off on my friend one day...
But also it's still a constant, on-going review and assessment of old habits for me. I have to check myself a lot. Sometimes I care too much about what other people think and so the pressure's on when I feel that she's "blown it" and need to show people I'm addressing it (like if she hits and pushes a kid or something). That's one of the bigger ones I have to let go of...
But yes, I am enjoying the unschool life very much and the easier *I* become, the easier and more likable it gets! = )
© 2015 Created by TheBananaGirl. Powered by


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Everybody was Kung Fu fighting
- Join Date
- Mar 2005
- Location
- Oxford, UK
- Posts
- 6,525
Posted On:
7/08/2005 3:56pm
Senior Member
- Join Date
- Aug 2004
- Location
- St. Petersburg, Fl
- Posts
- 1,044
Posted On:
7/08/2005 3:57pm
Style: Gracie Barra Jiu Jitsu--
nice butter fly, he looked frustrated for a while, god hes a beast. i thought you did good considereing giving up that much weight. most of your sweep attemps where killed by him posturing and droping that 100 extra pounds on you.
also looked like he almost got that anaconda on you got patients and escape.
Last edited by Bizzaro Root; 7/08/2005 4:00pm at .Eduardo "Why'd you stop."
Me "I was kicked in the head by the guys sparring next to me."
Eduardo "Ino what happened but i didnt say you could stop."
Me "Um.. I guess I keep going."
Eduardo "You dont stop until i say stop, you dont get tired until i say your tired, keep going."
Originally posted by Ralek
My cousin gave me some tapes of him doing tkd. I learned from those tapes. When I beat up an Akido instructor, and made him take rest breaks, I used TKD. I learned Bjj from watching ufc and pride and then I copied them and wrestled my cousin for practice. I choked him out and he tapped.
Registered Member
- Join Date
- Feb 2005
- Location
- Canada
- Posts
- 75
Posted On:
7/08/2005 4:26pm
Dang. I give you props for lasting that long agaisnt the guy - he'd have my ass for lunch, and I'm not 100 pounds lighter then him.There's no choice but to confront you, to engage you, to erase you. I've gone to great lengths to expand my threshold of pain. I will use my mistakes against you. There's no other choice.
Registered Member
- Join Date
- Jun 2005
- Posts
- 696
Posted On:
7/08/2005 6:49pm
Just a technical question, two actually:
Why'd you release the guard at the beginning, after you jumped into it?
Why did you go down on your back like that just after and let him come over top?
It looked delibrate I was just wondering what you were thinking.
Last edited by JKDChick; 7/08/2005 9:38pm at .Monkey Ninjas! Attack!
I blew up a frackin' star!
- Join Date
- May 2005
- Location
- Portland, OR
- Posts
- 87
Posted On:
7/08/2005 9:39pm--
Nice video.
One thing is on my mind though, so I might as well ask. I'm assuming that pulling guard like you did at the very beginning is something that you would only do in a competition... because otherwise, what would prevent your spine and the back of your head from being smashed to the ground (and could be quite dangerous without mats).
I probably answered my own question, so please disregard if this is so. Again, nice fight
Excellent work.
My critique:
Your first guard jump was perfect. The second one you got lazy. I'm sure you know this already though.
He had a nice Brabo in the beginning. Way to gut it out.
I liked the sit out attempts. The first one was close. You should have driven backwards a bit more and used your flexibility to spin in midair. You almost did it. The second one you didn't hook the arm and were slow.
When he tried to break your guard you should have gone after his ass with a vengence. I could see right there he was slowing down. The high guard was working. Just get a little more control of one arm. I suggest 2 on 1 with a guy that size. 1 on 1 he is just going to power right out of everything. The omoplata was good. If you feel like they can stand up out of it, try to scoot AWAY from them it breaks their base down where they can't post and allows you a moment to sit up.
When he backed out you shoulda got on his ass. Good recoveries though. Back to the butterfly guard. Consider an explosive drive forward sometimes that would knock him backwards. Probably the last thing he expects. Because honestly, you weren't gonna elevator that guy. Way too big for you.
Hahaha for the helicopter attempt! Big balls for you trying that. Next time get your head under there! Fucking awesome. But when he went for the headlock thing and had you buckled over you rolled when it LOOKED like 1 hard pull and you might have his back.
Ohhh you do try to drive him backward. He's just too big. Sucks.
Carefull with that gi-style guard sweep when he stands. He was slow in going for the calf slicer. You rolled well and came up strong. Don't give up on the single so easy. Drive! Drive! Drive!
Easy to sit back and say what you coulda/woulda/shoulda done though. I say good job. That guy wasn't too bad.
Registered Member
- Join Date
- Apr 2005
- Location
- Niagara Falls, NY
- Posts
- 229
Posted On:
7/08/2005 9:57pm
Style: MMA--
I'm not really a BJJ or all-grappling guy but i do freestyle wrestle, and ive noticed that for me if im wrestling a guy whos bigger than me, id rather counter them than meet them head on in a takedown battle so you really did the right thing trying to keep him in your guard rather than take him down with a shot or whatever.
perhaps u could have used the sprawl more? i dunno, theres some places where it seems like that would have helped. you did great though. he was a big dude and u by no means got destroyed.
Registered Member
- Join Date
- Jan 2005
- Posts
- 66
Posted On:
7/08/2005 10:22pm
Style: Taco Do--
Hey dude. You looked pretty good. Nice work against a guy with a very
strong base and some huge guns. Awesome stepping up your game with a dude that much bigger.
Nice butterfly guard work - you have a strong guard. I think most of the butterfly was pretty defensive - good defense by the way. I don't know how many elevators/sweeps are going to happen from there with a dude that big with that kind of base. The closest you got was working up into a high guard / semi rubber guard with that triangle -> arm bar -> omaplata he stood up out of. You might could have exploited that more. And gone more aggressive from the guard - you sat up twice and looked like you were going for the guillotine, but maybe string that and alternate two on ones going for an arm drag or isolating an arm to attack, constantly working. He had good posture, hard to attack. He was also over the top a lot with that front half nelson or whatever. Can't you block that when he's trying to sink it by bringing your elbow up and grabbing the back of your own neck same side? That would lead to the guard rather than being in trouble.
Overall, kick ass work. You've got a decent overall game. Muchos kudos from the armchair quarterback peanut gallery who you could tap 90% of.


text:
News about Festivals, including commentary and archival articles published in The New York Times.
Amy Winehouse Documentary Lets Nobody Off the Hook
Those who decided to make a film about the soul singer's legacy may not have gotten exactly what they expected, especially her family.
June 30, 2015, Tuesday
MORE ON FESTIVALS AND: Cannes International Film Festival , Winehouse, Amy , Documentary Films and Programs , Drug Abuse and Traffic , Music , Amy (Movie) , Kapadia, Asif
Wilco Tinkers and Experiments at a Festival It's Happy to Share
The Solid Sound Festival in North Adams, Mass., is determinedly upbeat, inclusive and family-friendly, and it features a convergence of grown-up indie tastes.
June 30, 2015, Tuesday
MORE ON FESTIVALS AND: Music , Wilco (Music Group) , Festivals , North Adams (Mass)
Glastonbury Festival Celebrates Its 45th Anniversary
Michael Eavis, the English dairy farmer who founded this vast, bucolic arts event, talks about its early days and the ingredients of its success.
June 27, 2015, Saturday
MORE ON FESTIVALS AND: Glastonbury Festival of Contemporary Performing Arts , Music
FarmBorough Festival, a Country Music Kickoff in the Big City
This weekend's three-day festival, starring Dierks Bentley, Brad Paisley and Luke Bryan, is a bet on New York's growing country market, from the company behind Governors Ball.
June 26, 2015, Friday
MORE ON FESTIVALS AND: Music , Festivals , New York City , Country Music , Bryan, Luke , Bentley, Dierks
Pop & Rock Listings for June 26-July 2
A selected guide to concerts in the New York City area.
June 26, 2015, Friday
MORE ON FESTIVALS AND: Music , Rock Music , Central Park Summerstage
Review: 'Poliuto,' a Rare Donizetti Bel Canto Thriller
This tale of religious persecution and martyrdom in Roman-occupied Armenia gets an updated staging at the Glyndebourne Festival.
June 24, 2015, Wednesday
MORE ON FESTIVALS AND: Opera , Glyndebourne Festival , Clement, Mariame (1974- ) , Donizetti, Gaetano , Fabiano, Michael , Martinez, Ana Marie , London Philharmonic , England , Golovatenko, Igor , Poliuto (Opera)
Review: Aston Magna and Boston Early Music Festivals Offer Diverse Monteverdi Experiences
In Great Barrington, Mass., the Boston Early Music Festival revived "Orfeo," in a stripped-down form, and the Aston Magna Music Festival performed the first program of its season, consisting almost entirely of Monteverdi madrigals.
June 24, 2015, Wednesday
MORE ON FESTIVALS AND: Boston Early Music Festival , Music
'Oklahoma!' Reimagined: Less Cornpone, but Fresh Cornbread
The director Daniel Fish's take on this classic includes an immersive element, but it also tries to stay true to the feel of the 1940s Rodgers and Hammerstein original.
June 23, 2015, Tuesday
MORE ON FESTIVALS AND: Theater , Fish, Daniel , Rodgers, Richard , Hammerstein, Oscar II , Bard SummerScape Festival , Testa, Mary , Heginbotham, John , Gray, Amber , Kluger, Daniel , Oklahoma! (Play)
Review: Aston Magna and Boston Early Music Festivals Offer Diverse Monteverdi Experiences
In Great Barrington, Mass., the Boston Early Music Festival revived "Orfeo," in a stripped-down form, and the Aston Magna Music Festival performed the first program of its season.
June 23, 2015, Tuesday
MORE ON FESTIVALS AND: Opera , Stepner, Daniel , Labelle, Dominique , L'Orfeo (Opera) , Boston Early Music Festival , Monteverdi, Claudio , Music , Festivals , Massachusetts , Blin, Gilbert
Cannes Lions Festival Shifts Focus Toward Ad Technology
Companies that had once operated on the fringes of the conference in France are now central to the event, which was created to showcase and award the best in advertising.
June 22, 2015, Monday
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text:
Customized TV Listings are available here: www.latimes.com/tvtimes
Click here to download TV listings for the week of July 6 - 12, 2014 in PDF format
Hell, yeah! A pro-wrestling legend invites all comers to try out his obstacle course in "Steve Austin's Broken Skull Challenge." 8 p.m. CMT
Not-so-innocent abroad: The concert special "Miley Cyrus: Bangerz Tour" finds pop music's enfant terrible taking the stage in Lisbon and Barcelona. 9 p.m. NBC
Julia Ormond, Jenna Dewan Tatum, Rachel Boston and Mädchen Amick are once again the "Witches of East End" on the return of this supernatural drama. 9 p.m. Lifetime
It's never too soon for nostalgia when Rob Lowe narrates a three-night survey of "The '90s: The Last Great Decade." 9 p.m. National Geographic Channel; also Mon., Tue.
All the world's a stage for the actor-activists of Belarus Free Theatre, as detailed in the 2013 doc "Dangerous Acts Starring the Unstable Elements of Belarus." 9 p.m. HBO
A disabled filmmaker turns his lens on some of the inspiring athletes who took part in 2012's Paralympic Games in a new documentary on "POV." 10 p.m. KOCE
"Ice Road Truckers" return for another season of trucking around on roads that are frankly far too icy for them to be trucking around on. 10 p.m. History Channel
FYI, Biography Channel is now FYI. Its first offering: "Married at First Sight" in which "Hello" is followed almost immediately by "I do." 6 p.m. FYI
"Time Scanners" take their high-tech gear to London to suss out the architectural secrets of St. Paul's Cathedral. 8 p.m. KOCE
The hit teen drama "Pretty Little Liars" hits the 100-episode mark. That's a whole lotta lies! 8 p.m. ABC Family
The 1944 disappearance of a plane carrying big-band leader Glenn Miller is probed on a new "History Detectives Special Investigations." 9 p.m. KOCE
My mother, my kidnapper: A teen (Kathryn Prescott) copes with a shocking revelation in the new drama "Finding Carter." 10 p.m. MTV
It's about to get "Fancy" up in here when Aussie chart-topper Iggy Azalea joins Jennifer Lopez, Nick Cannon and others at the "iHeartRadio Ultimate Pool Party." 8 p.m. KTLA


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text:
Novel autonomous replication sequences which are capable of maintaining plasmids as extrachromosomal elements in host strains of the genus Pichia are provided. In addition, novel constructs including these DNA sequences, as well as transformed organisms therewith are provided. Processes for producing the DNA sequences and constructs of the invention, as well as method for isolating such sequences from any source, are provided.
1. An isolated DNA fragment which comprises an autonomous replication sequence isolated from a strain of Pichia pastoris; which DNA fragment when inserted into a plasmid enables said plasmid to be maintained as an extrachromosomal element in multiple copies in a Pichia pastoris host, and which isolated DNA fragment when inserted into a plasmid causes increased frequency of transformation of a Pichia pastoris host compared to the frequency of transformation with plasmids which do not contain said isolated DNA fragment.
2. An isolated DNA fragment in accordance with claim 1 which has been isolated from Pichia pastoris NRRL Y-11430.
3. An isolated DNA fragment in accordance with claim 1 wherein said fragment has the nucleotide sequence: ##STR3##
4. An isolated DNA fragment in accordance with claim 1 wherein said fragment has the nucleotide sequence: ##STR4##
5. A hybrid plasmid capable of transforming a strain of Pichia pastoris which comprises an isolated DNA fragment in accordance with claim 1.
6. A hybrid plasmid capable of transforming a strain of Pichia pastoris which comprises the isolated DNA fragment of claim 3.
7. A hybrid plasmid capable of transforming a strain of Pichia pastoris which comprises the isolated DNA fragment of claim 4.
8. A hybrid plasmid in accordance with claim 5, which is pYJ30.
9. A hybrid plasmid in accordance with claim 5 which is pYJ32.
10. A strain of Pichia pastoris which has been transformed with the hybrid plasmid of claim 5.
11. A strain of Pichia pastoris which has been transformed with the hybrid plasmid of claim 8.
12. A strain of Pichia pastoris which has been transformed with the hybrid plasmid of claim 9.
13. Escherichia coli NRRL B-15890 (LE392-pYJ30).
14. Escherichia coli NRRL B-15891 (LE392-pYJ32).
15. A process for isolating DNA sequences from Pichia pastoris; wherein said DNA sequences have autonomous replication sequence activity in a transformable yeast host of the species Pichia pastoris; said process comprising:
(a) preparing a library of DNA fragments obtained from Pichia pastoris in a vector capable of transformation into a member of the species Pichia pastoris; wherein said vector comprises:
(i) a marker gene; wherein said marker gene comprises a functional gene which confers a selectable phenotype upon said yeast host,
(ii) bacterial sequences which enable transformation of bacteria with said vector, amplification of said vector in a bacterial host and selection for transformed bacterial host, but
(iii) substantially no yeast autonomous replication sequence activity;
(b) transforming said yeast host with said library; wherein said marker gene can be selected in said yeast host;
(c) collecting transformed colonies produced in step (b) and growing them under selective conditions;
(d) extracting total DNA from the cells which survive the selective growth conditions;
(e) transforming competent E. coli cells with the total DNA obtained in step (d);
(f) growing the transformed E. coli cells obtained in step (e) under selective growth conditions, wherein said selective growth conditions comprise media plus antibiotic to which said bacterial sequences provide resistance;
(g) recovering plasmid from said transformed E. coli cells;
(h) transforming said yeast host with the plasmid recovered in step (g);
(i) growing up the transformed yeast host obtained in step (h) under selective growth conditions;
(j) selecting and purifying colonies which grow well under the selective growth conditions of step (i);
(k) transferring the purified colonies to liquid culture and growing under selective growth conditions
(l) extracting total DNA from the cultures grown up in liquid culture;
(m) transforming competent E. coli cells with the DNA isolated in step (1);
(n) selecting those colonies in step (k) which gave the high transformation frequency of E. coli in step (m);
(o) recovering plasmid from those colonies selected in step (n); and
(p) removing the inserted DNA fragment contained in plasmid recovered in step (o).
16. A process in accordance with claim 15 wherein said host is auxotrophic mutant Pichia pastoris NRRL Y-15851 (GS115).
This invention relates to the field of recombinant DNA technology. In one of its aspects the invention relates to DNA fragments which are maintained as extrachromosomal elements in a host of the genus Pichia. In another aspect, the invention relates to expression vectors which incorporate the above-described DNA fragments. In yet another aspect, the invention relates to novel microorganisms transformed with the above-described expression vectors. In a further aspect, the invention relates to a process for isolating the novel DNA fragments of the invention.
The basic techniques employed in the field of recombinant DNA technology are known by those of skill in the art. The elements desirably present in order for a host mciroorganism to be useful for the practice of recombinant DNA technology include, but are not limited to:
(1) a gene encoding one or more desired polypeptide(s) and provided with adequate control sequences required for expression in the host microorganism,
(2) a vector, usually a plasmid, into which the gene with control sequences can be inserted. The vector serves to guarantee transfer of the gene into the cell and maintenance of DNA sequences in the cell. Where autonomous replication sequences are included in the vector, multicopies of the vector per cell can be obtained, as well as a high level of expression of the above-mentioned gene, and
(3) a suitable host mciroorganism into which the vector carrying the desired gene can be transformed, where the host microorganism also has the cellular apparatus to allow expression of the information coded for by the inserted gene.
A basic element employed in recombinant DNA technology is the plasmid, which is extrachromosomal, double-stranded DNA found in some microorganisms. Where plasmids have been found to naturally occur in microorganisms, they are often found to occur in multiple copies per cell. In addition to naturally occurring plasmids, a variety of man-made plasmids, or hybrid vectors, have been prepared. Included in the information encoded in plasmid DNA is that required to reproduce the plasmid in daughter cells, i.e., an autonomous replication sequence. One or more phenotypic selection characteristics must also be included in the information encoded in the plasmid DNA. The phenotypic selection characteristics permit clones of the host cell containing the plasmid of interest to be recognized and selected by preferential growth of the cells in selective media.
The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or restriction enzyme, each of which recognizes a specific, unique site on the plasmid DNA. Thereafter, homologous genes, heterologous genes, i.e., genes derived from organisms other than the host, or gene fragments may be inserted into the plasmid by endwise joining of the cleaved plasmid and desired genetic material at the cleavage site or at reconstructed ends adjacent to the cleavage site. The resulting recombined DNA material can be referred to as a hybrid vector.
DNA recombination is performed outside the host microorganism. The resulting hybrid vector can be introduced into the host microorganism by a process known as transformation. By growing the transformed microorganism, large quantities of the hybrid vector can be obtained. When the gene is properly inserted with reference to the portions of the plasmid which govern transcription and translation of the encoded DNA message, the resulting hybrid vector can be used to direct the production of the polypeptide sequence for which the inserted gene codes. The production of polypeptide in this fashion is referred to as gene expression.
Up to now, commercial efforts employing recombinant DNA technology for producing various polypeptides have centered on Escherichia coli as a host organism. However, in some situations E. coli may prove to be unsuitable as a host. For example, E. coli contains a number of toxic pyrogenic factors that must be eliminated from any polypeptide useful as a pharmaceutical product. The efficiency with which this purification can be achieved will, of course, vary with the particular polypeptide. In addition, the proteolytic activities of E. coli can seriously limit yields of some useful products. These and other considerations have led to increased interest in alternative hosts, in particular, the use of eukaryotic organisms for the production of polypeptide products is appealing.
The availability of means for the production of polypeptide products in eukaryotic systems, e.g., yeast, could provide significant advantages relative to the use of prokaryotic systems such as E. coli for the production of polypeptides encoded by recombinant DNA. Yeast has been employed in large scale fermentations for centuries, as compared to the relatively recent advent of large scale E. coli fermentations. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. In fact, growth of yeast such as Pichia pastoris to ultra-high cell densities, i.e., cell densities in excess of 100 g/L, is disclosed by Wegner in U.S. Pat. No. 4,414,329 (assigned to Phillips Petroleum Co.). Additional advantages of yeast hosts include the fact that many critical functions of the organism, e.g., oxidative phosphorylation, are located within organelles, and hence not exposed to the possible deleterious effects of the organism's production of polypeptides foreign to the wild-type host cells. As a eukaryotic organism, yeast may prove capable of glycosylating expressed polypeptide products where such glycosylation is important to the bioactivity of the polypeptide product. It is also possible that as a eukaryotic organism, yeast will exhibit the same codon preferences as higher organisms, thus tending toward more efficient production of expression products from mammalian genes or from complementary DNA (cDNA) obtained by reverse transcription from, for example, mammalian mRNA.
The development of poorly characterized yeast species as host/vector systems is severely hampered by the lack of knowledge about transformation conditions and suitable vectors. In addition, auxotrophic mutations are often not available, precluding a direct selection for transformants by auxotrophic complementation. If recombinant DNA technology is to fully sustain its promise, new host/vector systems must be devised which facilitate the manipulation of DNA as well as optimize expression of inserted DNA sequences so that the desired polypeptide products can be prepared under controlled conditions and in high yield.
An object of the invention is therefore novel autonomous replication sequences (ARS) which maintain plasmids as extrachromosomal elements in multi-copies per cell in hosts of the genus Pichia.
Another object of the invention is novel vectors capable of maintenance as extrachromosomal elements in hosts of the genus Pichia.
Yet another object of the invention is novel yeast strains of the genus Pichia.
These and other objects of the invention will become apparent from the disclosure and claims herein provided.
In accordance with the present invention, I have discovered, isolated and characterized autonomous replication sequences which aid the maintenance of recombinant DNA material as an extrachromosomal element in multi-copy per cell in host cells of the genus Pichia. In addition, a method is provided for isolating DNA sequences having autonomous replication activity in yeast of the genus Pichia from any source of DNA.
FIG. 1 is a restriction map of an autonomously replicating sequence of the invention (PARS1).
FIG. 2 is a restriction map of an autonomously replicating sequence of the invention (PARS2).
FIG. 3 is a restriction map of plasmid YEp13.
FIG. 4 is a restriction map of plasmid pYJ8.
FIG. 5 is a restriction map of plasmid pYJ8ΔCla.
FIG. 6 is a restriction map of plasmid pYM4.
FIG. 7 is a restriction map of plasmid pYJ30.
FIG. 8 is a restriction map of plasmid pYJ32.
FIG. 9 is a restriction map of plasmid pYA2.
The following abbreviations are used throughout this application to represent the restriction enzymes employed:
______________________________________Abbreviation Restriction enzyme______________________________________A AluIAh AhaIIIAv AvaIB BamHIB2 BglIIC ClaIH2 HindIIH3 HindIIIMb MboIINr NruIPs PstIPv2 PvuIIRs RsaIR1 EcoRIS SalISm SmaISp SphIS3 Sau3AIT TaqIXh XhoI______________________________________
In the attached figures, restriction sites employed for the manipulation of DNA but which are destroyed upon ligation are indicated by enclosing the abbreviation for the destroyed site in parenthesis. Restriction sites which are predicted by nucleic acid sequence data but have not been verified by actual restriction enzyme treatment are indicated by flagging the designated restriction site with an asterisk.
In accordance with the present invention, there are provided novel DNA fragments comprising autonomous replication sequences which maintain plasmids as extrachromosomal elements in hosts of the genus Pichia.
Further in accordance with the present invention, there is provided a process for isolating DNA sequences from any source which have autonomous replication properties in a host of the genus Pichia.
Host organisms contemplated to be useful for the practice of the present invention include the various species of the genus Pichia. One class of useful hosts are auxotrophic mutants, i.e., mutant strains which require supplementation with one or more amino acids, vitamins or other nutrients in order to grow. Transformation of such a mutant can be readily selected by employing, as part of the recombinant DNA material used to transform the mutant host, DNA sequences which code for the production of the missing gene product.
An especially preferred host yeast strain is the mutant Pichia pastoris GS115, which is a mutant defective in the ability to produce histidine, and has been identified as having the mutant genotype his4. GS115 was derived by mutagenesis of Pichia pastoris NRRL Y-11430 and has been deposited with the Northern Regional Research Center of the United States Department of Agriculture in Peoria, Ill., in order to ensure free access of the host to the public upon issuance of this application as a patent. Pichia pastoris GS115 has been assigned the accession number NRRL Y-15851, as of Aug. 31, 1984. This particular host is useful because it is an auxotrophic mutant deficient in the histidine pathway. It is, of course, readily recognized by those of skill in the art that mutants in many other genes important in Pichia metabolism also exist or can be isolated. Thus, many other hosts are possible for Pichia transformation, limited only by the availability of, or ability to isolate genes which code for the production of the gene product in which the mutant host is defective.
Pichia pastoris NRRL Y-15851 has been identified as a mutant defective in the production of histidinol dehydrogenase. This identification was accomplished by measuring the reduction of nicotinamide adenine dinucleotide (NAD) by a protein extract from cells of NRRL Y-15851 in the presence of the histidinol dehydrogenase substrate, histidinol. By analogy to the nomenclature employed with S. cerevisiae, the defect in NRRL Y-15851 is referred to as a his4C mutation.
The HIS4 gene was isolated from the strain P. pastoris NRRL Y-11430 by partial digestion of total chromosomal DNA with Sau3A followed by centrifugation through sucrose gradients. Fragments of 5 to 20 kbp were cloned into the BamHI cleavage site of the S. cerevisiae-E. coli shuttle vector YEp13 (ATCC 37115; FIG. 3) and transformed into E. coli. Approximately 50,000 colonies were selected and combined, then total plasmid DNA extracted. Spheroplasts of S. cerevisiae strain 5799-4D (NRRL Y-15859), a his4ABC mutant, were mixed with about 1 μg of the YEp13 Pichia DNA library by the procedure of Hinnen et al (1978) and allowed to regenerate in a medium deficient in histidine. The transformation resulted in about 1�103 prototrophic yeast colonies from a population of 5'107 total regenerable spheroplasts. A parallel control sample incubated without DNA produced no colonies. Total yeast DNA was extracted from 20 of the His+ colonies and transformed back into E. coli. Seventeen of the yeast DNA preparations produced ampicillin resistant colonies. These cloned fragments were further characterized by restriction enzyme sizing and mapping as well as by their ability to cross hybridize with a labelled S. cerevisiae HIS4 fragment at low stringency (post hybridization washes in 2xSSC at 55�). The HIS4-containing fragment each contained one or more fragments which hybridized to the S. cerevisiae HIS4 gene. One such HIS4-containing plasmid was recloned to give a HIS4-containing plasmid designated pYJ8 and is shown in FIG. 4. Plasmid pYJ8 contains pBR325 sequences, including functional chloramphenicol and ampicillin resistance genes, as well as the Pichia HIS4 gene.
By subcloning the 6.0 kbp Pichia DNA fragment from pYJ8, it was determined that a 2.7 kbp fragment of this DNA retained the ability to transform either Pichia or Saccharomyces strains deficient in HIS4A, HIS4B or HIS4C gene encoded activities. Thus, for example, Pichia pastoris NRRL Y-15851 (GS115), a his4 mutant, is able to grow on media without histidine supplementation when transformed with plasmids pYJ30 and pYJ32 (see FIGS. 7 and 8, respectively). These two plasmids, each of which contain the 2.7 kbp BglII fragment of Pichia chromosomal DNA, both encode the HIS4 gene function.
The experimental procedures for transformation of Pichia pastoris are presented in greater detail below (Example I). In order to develop a transformation system for P. pastoris, the auxotrophic mutant GS115 (NRRL Y-15851) was isolated and determined to be defective in the histidine pathway in that the strain has no detectable histidinol dehydrogenase activity.
GS115 (NRRL Y-15851) can be transformed by enzymatic digestion of the cell walls to give spheroplasts; the spheroplasts are then mixed with the transforming recombinant DNA material and incubated in the presence of calcium ions and polyethylene glycol, then regenerated in selective growth medium deficient in histidine. The transforming DNA includes the HIS4 gene in which the host strain is deficient, thus only transformed cells survive on the selective growth medium employed.
The vectors of the present invention contain Pichia-derived autonomous replication sequences (PARSs), which enhance both the transformation frequency of GS115 (NRRL Y-15851) and the maintenance of the vectors as stable extrachromosomal elements in yeasts of the genus Pichia. These autonomous replication sequences are useful because known yeast ARS elements isolated from S. cerevisiae do not function in hosts of the genus Pichia. Thus, in order to develop Pichia as an expression system useful for the production of polypeptide products in yeast, it became necessary to isolate DNA sequences with ARS activity in Pichia.
To search for Pichia ARSs, DNA from Pichia pastoris NRRL Y-15851 was partially digested with TaqI and 5 to 10 kbp fragments were isolated and cloned into the unique ClaI site of pYJ8ΔCla (See FIG. 5). Plasmid DNA was amplified in E. coli, recovered and used to transform Pichia pastoris NRRL Y-15851. Plasmid DNA was then recovered from about 10,000 His+Pichia colonies and used to retransform E. coli. Plasmids from about 10,000 ampicillin resistant E. coli colonies were isolated and then transformed back into P. pastoris NRRL Y-15851 (GS115; his4). Forty of the His+ yeast colonies from this sublibrary transformation were separately streaked onto selective medium and grown independently on the selective medium. Total yeast DNA was extracted from each of these 40 cultures and transformed into E. coli. Two plasmids, pYA63, containing PARS1 and pYA90 containing PARS2 were selected for further analysis. Both of these plasmids transformed Pichia pastoris NRRL Y-15851 (GS115) at very high frequency, were maintained as autonomous elements and each contained a novel fragment of P. pastoris DNA.
The novel autonomous replication sequences from pYA63 and pYA90 were subcloned into the unique ClaI site of plasmid pYM4 (see FIG. 6) to give plasmids pYJ30 and pYJ32, respectively. Plasmid pYJ30 is shown in detail in FIG. 7 while plasmid pYJ32 is similarly depicted in FIG. 8. The plasmids, transformed into an E. coli host, have been deposited with the Northern Regional Research Center of the U.S. Department of Agriculture, Peoria, Ill., to insure free access to the public upon issuance of this application as a patent. The deposited strains have been assigned accession numbers as follows:
______________________________________Plasmid Host Strain NRRL Accession No.______________________________________pYJ30 LE392 NRRL B-15890pYJ32 LE392 NRRL B-15891______________________________________
The autonomous replication sequences of the invention, PARS1 and PARS2, can conveniently be recovered from pYJ30 and pYJ32, respectively, by treating the plasmids with the restriction enzymes EcoRI and HindIII. The desired ARS element is then obtained with about 23 extra base pairs at the 5' end (adjacent to the R1 site) and with about 5 extra base pairs at the 3' end (adjacent the H3 site). The PARS1 and PARS2 inserts can, of course, also be recovered from pYJ30 and pYJ32 by treatment of the plasmids with a variety of other restriction enzymes, as readily recognized by those of skill in the art upon inspection of the restriction maps provided in FIGS. 7 and 8. The PARS1 and PARS2 inserts have been characterized by restriction enzyme mapping. These two DNA fragments are shown in FIGS. 1 and 2, respectively.
Due to the relatively small size of these DNA fragments, they have been completely sequenced. The nucleotide sequence for PARS1 has been determined to be: ##STR1##
The nucleotide sequence for PARS2 has been determined to be: ##STR2##
As a measure of the ability of the PARSs to maintain plasmids as autonomous elements in Pichia, cultures of yeast cells which had been transformed with plasmids pYJ30 and pYJ32 were grown in selective medium and periodically sampled. The state of the plasmid sequences in the cells was determined by Southern hybridization of unrestricted yeast DNAs to radioactively labelled pBR322. Plasmids pYJ30 and pYJ32 were maintained as autonomous elements in Pichia for at least 50 generations in the selective medium.
An average plasmid copy number per Pichia pastoris cell was derived from the ratio of the amount of the genomic copy of the Pichia HIS4 gene to that of plasmid-borne HIS4 gene. For Pichia pastoris cells transformed with pYA63 and pYJ30 (each containing PARS1) and pYA90 and pYJ32 (each containing PARS2), the average number of copies of plasmid DNA containing the Pichia HIS4 gene relative to the number of copies of Pichia chromosomal HIS4 gene was about 10-15. It is recognized by those of skill in the art that the values for copy number derived as described herein represent minimum estimates for the number of plasmid copies per cell.
In general, DNA sequences which have autonomous replication activity in a host of the genus Pichia can be isolated by transforming the Pichia host with a library of DNA fragments constructed in a vector which contains, among other DNA sequences, a marker gene, but does not contain any DNA sequences with ARS activity in Pichia. The marker gene employed will confer a selectable phenotype upon the host yeast strain. The frequency of transformation of the host strain with the vector will be increased by one or more orders of magnitude when DNA sequences with ARS activity are present in the vector compared to the frequency of transformation with unmodified vector. Thus, selection and isolation of transformed host will provide organisms carrying plasmids with inserted DNA sequences which have ARS activity. In this fashion, DNA sequences from any source which have ARS activity in Pichia can be readily isolated.
The buffers and solutions employed in the following examples have the composition given below:
______________________________________1 --M Tris buffer 121.1 g Tris base in 800 mL of H2 O; adjust pH to the desired value by adding concentrated (35%) aqueous HCl; allow solution to cool to room temperature before final pH adjustment; dilute to a final volume of 1L.TE buffer 1.0 m --M EDTA in 0.01 --M (pH 7.4) Tris bufferSSC 0.15 --M NaCl 15 m --M sodium citrate adjusted to pH 7.0 with NaOHDenhardts' Solution 5 g Ficoll(50�) 5 g polyvinylpyrrolidone 5 g Bovine serum albumin (BSA; Pentax Fraction V) brought to a total volume of 500 mL with waterLB (Luria-Bertani) 5 g Bacto-tryptoneMedium 5 g Bacto-yeast extract 2.5 g NaCl in 1 L of water, adjusted to pH 7.5 with NaOHYPD Medium 1% Bacto-yeast extract 2% Bacto-peptone 2% DextroseSD Medium 6.75 g yeast nitrogen base without amino acids (DIFCO) 2% Dextrose in 1 L of waterSED 1 --M Sorbitol 25 m --M EDTA 50 m --M DTTSCE Buffer 9.1 g Sorbitol 1.47 g Sodium citrate 0.168 g EDTA 50 mL H2 O pH to 5.8 with HClCaS 1 --M Sorbitol 10 m --M CaCl2 filter sterilizePEG Solution 20% polyethylene glycol-3350 10 m --M CaCl2 10 m --M Tris-HCl (pH 7.4) filter sterilizeSOS 1 --M Sorbitol 0.3 � YPD medium 10 m --M CaCl2______________________________________
The following abbreviations are used throughout the examples with the following meaning:
EDTA: ethylenediamine tetraacetic acid
SDS: sodium dodecyl sulfate
DTT: dithiothreitol
Several procedures carried out on a routine basis follow a standard protocol which will be detailed here.
Centrifugation is carried out for a period of time and at a spin rate sufficient to provide a clear supernatant. Generally, centrifugation of yeast cells is carried out at at least 1500 g for at least 5 minutes.
Nucleic acid extractions with phenol/chloroform/isoamyl alcohol involve contacting the nucleic acid containing solution with an equal volume of a 50:48:2 ratio by volume mixture of phenol, chloroform and isoamyl alcohol, respectively. Extractions with chloroform/isoamyl alcohol involve contacting the solution to be treated with an equal volume of 48:2 ratio by volume mixture of chloroform and isoamyl alcohol.
When gels, filters, etc. are described as being washed or soaked in a specified solution, the entire gel, filter, or the like was immersed in an appropriate vessel (pan, dish, vial, etc.) in order to contact the entire surface of the gel, filter, or the like with the solution of interest.
Ethanol precipitation of nucleic acids involves first adjusting the salt content of the nucleic acid-containing solution, if necessary, then contacting the solution with two volumes of cold ethanol, then collecting the precipitate by centrifugation.
A. Cell Growth
1. Inoculate a colony of Pichia pastoris GS115 (NRRL Y-15851) into about 10 mL of YPD medium and shake culture at 30� C. for 12-20 hrs.
2. After about 12-20 hrs., dilute cells to an OD600 of about 0.01-0.1 and maintain cells in log growth phase in YPD medium at 30� C. for about 6-8 hrs.
3. After about 6-8 hrs, inoculate 100 mL of YPD medium with 0.5 mL of the seed culture at an OD600 of about 0.1 (or equivalent amount). Shake at 30� C. for about 12-20 hrs.
4. Harvest culture when OD600 is about 0.2-0.3 (after approximately 16-20 hrs) by centrifugation at 1500 g for 5 minutes.
B. Preparation of Spheroplasts
1. Wash cells once in 10 mL of sterile water. (All centrifugations for steps 1-5 are at 1500 g for 5 minutes.)
2. Wash cells once in 10 mL of freshly prepared SED.
3. Wash cells twice in 10 mL of sterile 1 M Sorbitol.
4. Resuspend cells in 10 mL SCE buffer.
5. Add 5-10 μL of 4 mg/mL Zymolyase 60,000 (Miles Laboratories). Incubate cells at 30� C. for about 30-60 minutes.
Since the preparation of spheroplasts is a critical step in the transformation procedure, one should monitor spheroplast formation as follows: add 100 μL aliquots of cells to 900 μL of 5% SDS and 900 μL of 1 M Sorbitol before or just after the addition of zymolyase and at various times during the incubation period. Stop the incubation at the point where cells lyse in SDS but not in sorbitol (usually between 30 and 60 minutes of incubation).
6. Wash spheroplasts twice in 10 mL of sterile 1 M Sorbitol by centrifugation at 1000 g for 5-10 minutes. (The time and speed for centrifugation may vary; centrifuge enough to pellet spheroplasts but not so much that they rupture from the force.)
7. Wash cells once in 10 mL of sterile CaS.
8. Resuspend cells in total of 0.6 mL of CaS.
C. Transformation
1. Add DNA samples (up to 20 μL volume) to 12�75 mm sterile polypropylene tubes. (DNA should be in water or TE buffer; for maximum transformation frequencies with small amounts of DNA, it is advisable to add about 1 μL of 5 mg/mL sonicated E. coli DNA to each sample.)
2. Add 100 μL of spheroplasts to each DNA sample and incubate at room temperature for about 20 minutes.
3. Add 1 mL of PEG solution to each sample and incubate at room temperature for about 15 minutes.
4. Centrifuge samples at 1000 g for 5-10 minutes and decant PEG solution.
5. Resuspend samples in 150 μL of SOS and incubate for 30 minutes at room temperature.
6. Add 850 μL of sterile 1 M Sorbitol and plate aliquots of samples as described below.
D. Regeneration of Spheroplasts
1. Recipe for Regeneration Agar Medium:
a. Agar-Sorbitol- 9 g Bacto-agar, 54.6 g Sorbitol, 240 mL H2 O, autoclave.
b. 10X Glucoseo- 20 g Dextrose, 100 mL H2 O, autoclave.
c. 10X SC- 6.75 g Yeast Nitrogen Base without amino acids, 100 mL H2 O, autoclave. (Add any desired amino acid or nucleic acid up to a concentration of 200 μg/mL before or after autoclaving.)
d. Add 30 mL of 10X Glucose and 30 mL of 10X SC to 300 mL of the melted Agar-Sorbitol solution. Add 0.6 mL of 0.2 mg/mL biotin and any other desired amino acid or nucleic acid to a concentration of 20 μg/mL. Hold melted Regeneration Agar at 55�-60� C.
2. Plating of Transformation Samples:
Pour bottom agar layer of 10 mL Regeneration Agar per plate at least 30 minutes before transformation samples are ready. Distribute 10 mL aliquots of Regeneration Agar to tubes in a 45�-50� C. bath during the period that transformation samples are in SOS. Add aliquots of transformation samples described above to tubes with Regeneration Agar and pour onto bottom agar layer of plates. Add a quantity of each sample to 10 mL aliquots of melted Regeneration Agar held at 45�-50� C. and pour each onto plates containing a solid 10 mL bottom agar layer of Regeneration Agar.
3. Determination of Quality of Spheroplast Preparation:
Remove 10 μL of one sample and dilute 100 times by addition to 990 μL of 1 M Sorbitol. Remove 10 μL of the 100 fold dilution and dilute an additional 100 times by addition to a second 990 μL aliquot of 1 M Sorbitol. Spread plate 100 μL of both dilutions on YPD agar medium to determine the concentration of unspheroplasted whole cells remaining in the preparation. Add 100 μL of each dilution to 10 mL of Regeneration Agar supplemented with 40 μg/mL histidine to determine total regeneratable spheroplasts. Good values for a transformation experiment are 1-3�107 total regeneratable spheroplasts/mL and about 1�103 whole cells/mL.
4. Incubate plates at 30� C. for 3-5 days.
A. Strains
The strains employed include:
(a) Pichia pastoris strain NRRL Y-11430;
(b) Pichia pastoris NRRL Y-15851 (GS115-his4); and
(c) E. coli strain 848 (F- met thi gal T1 R φ80S hsdR- hsdM+).
B. Plasmids
pYA2 (FIG. 9) which consists of the S. cerevisiae HIS4 gene on a 9.3 kb PstI fragment inserted at the PstI site of pBR325 was the source of the S. cerevisiae HIS4 gene fragments and has been deposited in an E. coli host and is available to the public as NRRL B-15874.
pYJ8ΔCla (FIG. 5), which is a derivative of pYJ8 (FIG. 4), was created by ClaI digestion of pYJ8 and ligation.
C. Media
Pichia pastoris was grown in YPD (rich) or IMG (minimal) media. IMG, a minimal medium, consists of the following:
1. IM1 Salts at a final concentration of 36.7 mM KH2 PO4, 22.7 mM (NH4)2 SO4, 2.0 mM MgSO4 μ7H2 O, 6.7 mM KCl, 0.7 mM CaCl2.2H2 O, prepared as a 10x stock solution and autoclaved;
2. Trace Salts at a final concentration of 0.2 μM CuSo4.5H2 O, 1.25 μM KI, 4.5 μM MnSO4 μH2 O, 2.0 μM NaMoO4 μ2H2 O, 0.75 μM H3 BO3, 17.5 μM ZnSO4.7H2 O, 44.5 μM FeCl3.6H2 O, prepared as a 400x stock solution and filter sterilized;
3. 0.4 μg/mL biotin; and
4. 2% dextrose.
E. coli was cultured in either LB medium or 2B medium (0.2% NH4 PO4, 1.2% Na2 HPO4, 0.013% MgSO4.7H2 O, 0.074% CaCl2.2H2 O, 1 μg/mL thiamine and 0.4% dextrose) supplemented with 100 μg/mL tryptophan, and 0.2% Casamino acids.
D. DNA Isolations
1. Large Scale Preparations of Yeast DNA:
Both Pichia pastoris and S. cerevisiae DNA preparations were carried out by growing yeast cells in 100 mL of minimal medium until A600 equals 1-2 and then harvesting the cells by centrifugation at 2,000 g for 5 minutes. The cells were washed once in H2 O, once in SED, once in 1 M sorbitol and then suspended in 5 mL of 0.1 M Tris-HCl (pH 7.0) which is 1 M in sorbitol. The cells were mixed with 50-100 μL of a 4 mg/mL solution of Zymolase 60,000 (Miles Laboratories) and incubated at 30� C. for 1 hour to digest the cell walls. The spheroplast preparation was then centrifuged at 1000 g for 5-10 minutes and suspended in Lysis buffer (0.1% SDS, 10 mM Tris-HCl, (pH 7.4), 5 mM EDTA and 50 mM NaCl). Proteinase K (Boehringer Mannheim) and RNase A (Sigma) were each added to 100 μg/mL and the mixtures incubated at 37� C. for 30 minutes. DNA was deproteinized by gently mixing the preparation with an equal volume of chloroform containing isoamyl alcohol and the phases were separated by centrifugation at 12,0000 g for 20 minutes. The upper (aqueous) phase was drawn off into a fresh tube and extracted with an equal volume of phenol/ chloroform/isoamyl alcohol. The phases were separated as before and the top phase placed in a tube containing 2-3 volumes of cold 100% ethanol. The sample was gently mixed and DNA was collected by spooling onto a plastic rod. The DNA was immediately dissolved in 1 mL of TE buffer and dialyzed overnight at 4� C. against 100 volumes TE buffer.
2. Small Scale Yeast DNA Preparations:
Five mL of yeast cultures in minimal medium were grown until A600 equals 1-5 and harvested by centrifugation at 2,000 g for 5 minutes. Cells were suspended in 1 mL of SED and transferred to a 1.5 mL microfuge tube, washed once in 1 M sorbitol and resuspended in 0.5 mL of 0.1 M Tris-HCl (pH 7.4) which is 1 M sorbitol. Zymolyase 60,000 (Miles Laboratories; 10 μL of a 4 mg/mL solution) was added to each sample and the cells were incubated for 30-60 minutes at 30� C. Cells were then centrifuged for 1 minute, suspended in the Lysis buffer and incubated at 65�-70� C. After 15 minutes the samples were mixed with 100 μL of 5 M potassium acetate, held in an ice bath for 15 minutes and centrifuged for 5 minutes. The supernatants were decanted into a fresh microfuge tube containing 1 mL of 100% ethanol, mixed and immediately centrifuged for 10 seconds. Finally, the DNA pellets were air dried for 10-15 minutes and dissolved in 50 μL of TE buffer.
3. Large Scale E. coli DNA Isolations:
E. coli cultures for large scale (0.5-1 L) plasmid preparations were grown at 37� C. with shaking in 2B medium supplemented as described above and with the appropriate antibiotic. For cells which contained pBR322 derived plasmids, cultures were grown to an A550 of about 0.7 at which time sufficient chloramphenicol was added to give a concentration of 100 μg/mL and cells harvested approximately 15 hours later. Strains which contained pBR325 derived plasmids were inoculated into the supplemented 2B medium at a starting A550 of about 0.01-0.05 and incubated with shaking at 37� C. for 20-24 hours before harvesting. Plasmids were isolated by the alkaline lysis method described by Birnboim and Doly (1979).
4. Small Scale E. coli DNA Preparations:
For small scale rapid plasmid isolations, 2 mL cultures in the supplemented 2B medium with antibiotic were grown overnight at 37� C. with shaking and harvested by centrifugation in 1.5 mL microfuge tubes. Plasmids were isolated by the alkaline lysis method described by Birnboim and Doly (1979). E. Restriction of DNA and Fragment Isolation
Restriction enzymes were obtained from New England Biolabs and Bethesda Research Laboratories and digestions were performed by routine techniques. Restriction mappings were carried out by comparing parallel digestions of plasmids with and without insert DNA. Restriction fragments were purified by electroelution from agarose gels into Whatman 3 MM paper strips backed by dialysis tubing. The fragments were recovered from the paper and tubing by 3-4 washings with 0.1-0.2 mL volumes of a solution which contained 0.1 M NaCl, 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA. Finally, the fragments were extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol and redissolved in a small volume of TE buffer.
F. P. pastoris Autonomous Replication Sequence Library
Construction in E. coli.
For the Pichia pastoris DNA-pYJ8ΔCla library construction, 50 μg of pYJ8≢Cla was digested to completion with ClaI and treated with calf intestinal alkaline phsphatase to remove the terminal 5' phosphate from the DNA. A 100 μg aliquot of DNA from Pichia pastoris NRRL Y-15851 was partially digested with 20 units of TaqI by incubation for 5 minutes at 65� C. in a total volume of 1 mL. Fragments of 5 to 10 kbp were size selected by electroelution from a 0.5% agarose gel (See Example II, Section E). One μg of the vector and 2 μg of the Pichia TaqI fragments were mixed with 20 units of T4 DNA ligase (Bethesda Research Laboratories) in a total volume of 200 μL and incubated overnight at 4� C. The ligated DNAs were transformed into E. coli by adding the entire ligation reaction mix to 2 mL of competent E. coli 848 cells and incubating for 15 minutes at 0� C. The mixture was warmed to 37� C. for 5 minutes after which time 40 mL of LB medium was added and the 37� C. incubation continued for another 1 hour. Ampicillin was then added to give a total concentration of 100 μg/mL and the incubation continued for a second hour. Finally, the cells were centrifuged for 10 minutes at 3,000 g, resuspended in 1 mL of fresh LB medium and spread in equal aliquots on 10 LB agar plates containing 100 μg/mL of ampicillin. The approximately 10,000 colonies which resulted were scraped from the plates and a portion of the cells was inoculated into 500 mL of the supplemented 2B medium at a starting A550 of about 0.1. The culture was grown and plasmid was extracted as described above.
G. Southern Hybridizations
Hybridizations were carried out by the method described by Southern (1975). For transfer of large or supercoiled DNA molecules to nitrocellulose, DNA was first partially hydrolyzed by soaking agarose gels in 0.25 M HCl for 10 minutes prior to alkali denaturation. The hybridization of labelled fragments from the S. cerevisiae HIS4 gene to Pichia pastoris DNA was performed in the presence of 50% formamide, 6x SSC 5x Denhardt's, 0.1% SDS, 1 mM EDTA, and 100 μg/mL denatured herring sperm DNA at 42� C. Post-hybridization washes were in 2x SSC, 1 mM EDTA, 0.1% SDS and 1.0% sodium pyrophosphate at 55� C.
H. 32 P-labelling
Nick translation was performed by the method described by Rigby et al (1977).
I. DNA Sequencing
DNA sequencing was by the dideoxynucleotide chain termination method of Sanger et al (1980). J. Isolation of Pichia Autonomous Replication Sequences
The Pichia library constructed as described in Section F, above, was used to transform Pichia pastoris NRRL Y-15851. Plasmid DNA was amplified in E. coli, recovered and used to transform Pichia pastoris NRRL Y-15851. Plasmid DNA was then recovered from about 10,000 His+Pichia colonies and used to retransform E. coli. Plasmids from about 10,000 ampicillin resistant E. coli colonies were isolated and then transformed back into P. pastoris NRRL Y-15851 (GS115; his4). Forty of the His+yeast colonies from this sublibrary transformation were separately streaked onto selective medium and grown independently on the selective medium. Total yeast DNA was extracted from each of these 40 cultures and transformed into E. coli. Two plasmids, pYA63, containing PARS1 and pYA90 containing PARS2 were selected for further analysis. Both of these plasmids transformed Pichia pastoris NRRL Y-15851 (GS115) at very high frequency, were maintained as autonomous elements and each contained a novel fragment of P. pastoris DNA.
K. Analysis of Pichia pastoris Transformants for Autonomous
Replication Sequences
The ability of Pichia ARS-containing plasmids to be maintained as autonomous elements in Pichia pastoris cells was determined as follows: A transformant colony was picked from the regeneration agar plate and streaked onto an SD medium agar plate and inoculated into liquid IMG medium. The SD plate was incubated at 30� C. for 3 days after which time a single colony was picked from this plate, streaked onto a second SD plate and inoculated into a second flask of IMG medium. This process was repeated a third time. The 3 IMG cultures were grown at 30� C. with shaking to an A600 of about 1-2 and then harvested by centrifugation. DNA from the yeast cultures was extracted as described above, electrophoresed at 30 Volts and 30 mAmps for 10-15 hours into 0.8% agarose gels, transferred to nitrocellulose and hybridized to 32 P-labelled pBR322 or pBR325 as described above. As controls, a sample containing 10 ng of plasmid isolated from E. coli and a sample containing 1-2 μg of untransformed Pichia pastoris NRRL Y-15851 (GS115) DNA were electrophoresed in parallel with the experimental samples.
In each of the Pichia PARS-containing plasmid transformants examined, the labelled probe hybridized in a pattern identical to the plasmid DNA isolated from E. coli. As a control, the labelled probe was found to hybridize to high molecular weight chromosomal DNA from Pichia pastoris NRRL Y-15851 (GS115) when transformed with pYJ8ΔCla (an integrative transforming vector which has no ARS activity in Pichia). Probe did not hybridize to DNA from untransformed NRRL Y-15851.
As a more quantitative measure of the ability of the PARSs to maintain plasmids as autonomous elements in Pichia, cultures of yeast cells which had been transformed with plasmids pYJ30 and pYJ32 were grown in selective medium and periodically sampled. The state of the plasmid sequences in the cells was determined by Southern hybridization of unrestricted yeast DNAs to radioactively labelled pBR322. Plasmids pYJ30 and pYJ32 were maintained as autonomous elements in Pichia for at least 50 generations in the selective medium.
L. Plasmid Copy Number Determination
An average plasmid copy number per P. pastoris cell was derived from the ratio of the amount of the genomic copy of the P. pastoris HIS4 gene to that of a plasmid-borne HIS4 gene. Since the strains examined contained plasmids with the Pichia HIS4 gene, DNAs were extracted, digested with restriction endonucleases, electrophoresed into an agarose gel, transferred to a nitrocellulose filter and hybridized with a 32 P-labelled 2.7 kbp BglII fragment containing the Pichia HIS4 gene. After post-hybridization washing, a series of X-ray films were exposed to the filter for specific lengths of time and scanned on a Beckman DU-8B spectrophotometer which was programmed with a Compuset Module for slab gels. Results are summarized in the Table.
TABLE______________________________________Characterization of PARS-Containing Pichiapastoris Transformants Autonomous Generations as Replication Reference Autonomous CopyPlasmid Sequence Figure Element Number______________________________________pYM4 -- 6 -- --pYJ30 PARS1 7 50 13pYJ32 PARS2 8 50 13______________________________________
Birnboim and Doly (1979) Nucl. Acis res. 7, 1513-1523.
Hinnen et al (1978) Proc. Nat. Acad. Sci., USA 75, 1929-1933.
Rigby et al (1977) J. Mol. Biol. 113, 237.
Southern (1975) J. Mol. Biol. 98 503-517. Sanger et al (1980) J. mol. Biol. 143, 161-178.
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Isringhausen Imports
(217) 528-2277
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Customer Reviews (2 reviews)2 Sort by: Date | Helpful
When I brought the car in the last time, your technician he told you that it was this sensor unit on my tranmission, so we had it replaced to the tune of $3700 all in.
I realize that they also did the shocks and struts during that time. Should I be concerned that the wheels or bearing now may be @ risk now as well.
Do you have my old CD unit back from the salvage folks yet? I would like to be able to collect me CD's that have been out now for at least eight months
As a result of this recent poor quality of service, I am going to be informing the US Better Business Bureau regarding the poor overall quality of craftsmenship received @ your establishment.
That may not mean anything to you, personally, but it will be on record w/ the Federal Govenement and it will be available to others, who need to be aware of the lack of customer care that your facility should be noted for and the poor quality of mechanical service. This is the the fifth time you have had this car in your care and have never corrected the problems that your technician determined to be root of the problem / deficiency. Your organization had no problem taking my money, but they continuosly fail to fix the problem /deficieny
AS to regard to the inferior workmanship on the braking system of my BMW, which put my life, as well as the lives of others @ risk.
I have retained all records related the maintenance performed @ your service center and you will be hearing from my attorneys soon.


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Tel: (619)443-1440
Our Lady Of Perpetual Help School proudly serves students throughout the East County region of San Diego including Lakeside, Santee, El Cajon, Alpine, Ramona, Pine Valley, and the Native American Communities of this area.
Our Lady of Perpetual Help School is a Christ-centered, child focused Catholic elementary school celebrating over forty years of serving the educational needs of students in the East County of the Diocese of San Diego.
Our educational program is directed toward the growth of the whole person so that each student may become a fully integrated human being and thus lead to a fuller understanding and appreciation of life in Christ, His Church and in society at large.
In recognition that parents are the primary educators of their children, the role of the school community is to support, enhance and complement.
Teachers serve not only as dedicated and qualified instructors, but they must lead in the creation of a Christian community in which the message of Christ is proclaimed in words and witness.
The mission of Our Lady of Perpetual Help School is to empower students to be spiritually, academically, socially and culturally prepared to meet the challenges of life.
OLPH School seeks to guide the religious development of each student through evangelization, catechesis, and religious instruction.
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Conductance is an expression of the ease with which electric current flows through a substance.
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Conductance is an expression of the ease with which electric current flows through a substance. In equations, conductance is symbolized by the uppercase letter G. The standard unit of conductance is the siemens (abbreviated S), formerly known as the mho.
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When a current of one ampere (1 A) passes through a component across which a voltage of one volt (1 V) exists, then the conductance of that component is 1 S. The siemens is, in fact, equivalent to one ampere per volt. If G is the conductance of a component (in siemens), I is the current through the component (in amperes), and E is the voltage across the component (in volts), then:
In general, when the applied voltage is held constant, the current in a direct-current (DC) circuit is directly proportional to the conductance. If the conductance is doubled, the current is also doubled; if the conductance is cut to 1/10 its initial value, the current also becomes 1/10 as great. This rule also holds for most low-frequency alternating-current (AC) systems, such as household utility circuits. In some AC circuits, especially at high frequencies, the situation is more complex, because some components in these systems store and release energy, as well as dissipating or converting it.
Conductance is inversely related to resistance. If R is the resistance of a component or device (in ohms), then the conductance G (in siemens) is given by:
'conductance' is part of the:
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text: KANSAS CITY, Mo. -- Rookie De'Anthony Thomas hasn't been a constant at Kansas City Chiefs' offseason practice, which concludes Thursday. He had to skip 10 of their 16 practices because of NFL rules prohibiting players from participating while their college was still in session, as Oregon was until last week.


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Home / Academic Programs / Arts / Conservatory of Theatre Arts / Purchase Rep
Purchase Repertory Theatre
"Purchase Rep … continues to present classic and contemporary works with production values that eclipse those on Broadway. And the talent, whose work is Broadway-bound … make the air in the black-box theater crackle with possibilities."
~ Peter D. Kramer, The Journal News & LoHud.com
Purchase Repertory Theatre 2014–15 Season
Fall 2014:
Tales from the Vienna Woods by Ödön von Horváth: Oct. 10–18
Directed by David Bassuk
Marat/Sade by Peter Weiss: Oct. 31–Nov. 8
Directed by A. Dean Irby
Mud, River, Stone by Lynn Nottage: Dec. 5–13
Directed by Joyce Sylvester
Rapture by Jeanne Marshall: Dec. 6–13
Directed by Jane Mandel
Spring 2015:
The Green Bird by Carlo Gozzi: Feb. 6–14
Directed by Mace Perlman
Romeo and Juliet by William Shakespeare: March 13–21
Directed by Chris McCann
Stage Door by Edna Ferber and George S. Kaufman: April 17–25
Directed by Sarah Grace Wilson
For Schedule, Tickets, and Season Passes:
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Game one of the doubleheader on Saturday between Florida & No. 4 LSU at McKethan Stadium has been pushed back to a 4 p.m. start.
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Thomas R. Webster, Ph.D.
Dean, School of Communication and Performing Arts
What a magnanimous pleasure it is to have a place of service at ETBU. Our School of Communication and Performing Arts is an active and thriving place where students grow and faculty motivate and encourage. Each academic year brings new opportunities and exciting concerts and productions. I am delighted that you have found our online home, and I hope that you will soon visit us on campus. See the information below and the online calendar of events to select the best time to visit. We are anxious to see you at one of our upcoming events. Feel free to contact me for any reason if we can be of service to you. Come see for yourself how we are "Engaging Minds, Embracing Faith, and Empowering Leaders."
TO PROSPECTIVE STUDENTS: I invite you set up your campus visit audition today. Once you meet our faculty and students and see for yourself the quality of the education we offer, I am confident that you will choose ETBU as your college home. During your visit you will also meet the individuals who can help you make your college dreams come true. Our enrollment management staff will guide you through the admissions and financial aid process. The personal attention you receive through the process will begin from your initial contact with us and continue throughout your college experience and into your professional life as a proud ETBU graduate. Come see us soon, and give us the opportunity to show you why ETBU should be your college home.
TO ARTS PATRONS: I hope you will join us for our next performance event. You will be delighted at the exceptional quality of the performances and the outstanding talent of our faculty and students. ETBU offers many performances events each semester, and you are sure to find many that you would enjoy. Our Department of Music presents performances by our choral and band programs, chamber music concerts, and several faculty and guest artist recitals. Our Department of Theatre Arts presents a wide array of productions each year, and our two departments combine forces each year to present either a large-scale musical theatre production or an opera performance. We utilize many performance venues on campus and throughout the community, so read the event calendar carefully. Feel free to contact the School of Communication and Performing Arts Office at 903-923-2158 if you have any questions. I look forward to seeing you very soon.
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Third of three hand lettered love notes, inspired by Italian Film. This is my personal favourite, and a style I'd like to further explore. As the other two are, this is dedicated to my wonderful girlfriend – and soon to be more – Rubie.
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Brenton Williams scored 24 points for South Carolina, and Kentucky coach John Calipari was ejected midway through the second half as the Gamecocks held off the 17th-ranked Wildcats 72-67 on Saturday night, South Carolina (11-18, 4-12 Southeastern Conference) led by three at halftime and hit six of its first eight shots in the second half to go up 48-32 with 14:13 left. Kentucky made a furious charge after Calipari was tossed, but came up short. The Wildcats (21-8, 11-5) couldn't make a basket, going 15:38 with only one field goal - on a goaltending call. Calipari was ejected with his team down 12 after getting a second technical with 10:21 left. He walked out of the arena with his suit coat buttoned but his shirttail out.
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Kentucky Falls at South Carolina, 72-67
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Three paratroopers on Asad Airbase, Iraq
Three paratroopers with 1st Brigade, 82nd Airborne Division (Advise and Assist), move off a drop zone near Al Asad Airbase, Iraq, Feb. 12, as part of a training exercise they hope will lead to combined U.S. – Iraqi training jumps and an enduring strategic partnership. For security, an Apache helicopter flew along the drop zone perimeter throughout the training exercise.
See more at www.army.mil
Paratroopers prep for U.S-Iraqi combined training
We proud of yew
Determined though scared, I walk my beat
On the deadly streets of Baghdad.
Searching for any who plot our harm
Or by our death are joyous and glad.
Standing in shadows caused by the moon
I'm reminded of my nights back home.
I wonder if the woman I love
Is growing tired of sleeping alone?
I feel remorse for all who live here
For this place is a madman's hell.
And those who wish to keep it that way
Must be killed or locked away in jail.
My greatest fear is not my death
But that I'll end up in a wheelchair.
Disabled for the rest of my life,
Depending on others for my care.
My wife, she prays for my safe return
As night and day more GI's are killed.
She knows quite well, whatever it takes
The oath I've given will be fulfilled.
The king of Baghdad has fallen
Never to dictate again.
Man shall sentence him for this crimes
And heaven shun him for his sin.
For his tyranny, he was famous
In every capital on earth.
Till apprehended in his spider hole
Completely stripped of his worth.
He is guilty of rape and genocide
While he ruled without remorse.
His power and prestige were toppled
Once George Bush set his course.
Though it may seem that the wicked triumph
And have conquered by their brutality of hand,
Through the power of faith they are defeated
By the seed of goodness in man.
America is the birthday cake of earth
As the ants march from every direction.
Thank God for all who have sworn to defend her
Serving with love, honor, pride, and affection.
Since the first day George Washington marched off to war
There have been those who have wished our demise.
Their hatred, fueled by jealousy and greed
Was defeated by our brave and the wise.
Once again, we must face a formidable foe
Who have pledged by their God to destroy us all
Misusing their faith as an excuse to kill
As for a worldwide jihad, their leaders call.
Some say we should try to appease them
For if we resist, they'll hate us even more.
But the David's among us shall cast our stones
Defeating them, as it was done before.
They serve to preserve our forefathers dreams,
Prayers, visions and determination.
Risking all in pursuit of fulfillment of duty
To God, freedom, faith, honor and nation.
Despite dismemberment, death and loneliness
Patriots enlist to safeguard our flag.
With honor, faith, purpose and courage
They battle the sadistic that brag.
Throughout man's past as a creature of earth
War has always plagued his expectance.
Greed, hate, fear, envy and rage
Have overruled rapture and repentance.
David was a soldier who lived by his faith
Which gave him the will to become brave.
He stood up to terror and toppled the giant
Leaving Goliath headless and alone in his grave.
David's call thrives in hearts of soldiers today
Shielding liberty from the warmongers of hell.
Facing down evil refusing to summit
Ensuring freedom and justice are alive and well.
By Soldier For The Lord
Tom Zart
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Taken on February 12, 2010
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