text:
|Property type
|Minimum Stay
|4 - 7 nights
Our beautiful newly furnished luxury 4 bedroom/3 Bath Condo is finest located at Regency @ Poipu Kai Resort. It is one of only a handful of air-conditioned properties on the Island of Kauai. Our Condo is on the 2nd & 3rd floor with a fantastic view of the mountain range. The resort is located just a short stroll to several world class beaches including Shipwreck Beach, Brenneckes Beach and Poipu Beach which makes them the perfect location for any size group or family. If you have younger children along for the trip you won't find a more perfect children's beach than the little beach formed between Brenneckes and Poipu beaches. Protected from the surf by a natural rock outcropping and featuring a very shallow and gently sloping beach, the children will have a blast!
Excellent service dealing with Jennifer to make the arrangements. She was very helpful and honest, quick to respond to questions, and exceeded our expectations leading up to our trip. Everything was as expected, there were no unwelcome surprises. Also appreciated the great driving directions from Airport since we arrived after dark.
The condo was what we were lead to believe: great location, clean, well equipped with very good quality. The kitchen disposal broke down on day 2, and the on site host had a plumber there the next morning to fix it. Good quality and plenty of beach supplies was much appreciated - we didn't have to bring anything.
Location was excellent. A beautiful 10 minute walk on path to Poipu Beach (best beach on the island). 5 minute drive to grocery store in Koloa. 10 minute walk on path east to the Hyatt. Beautiful view out of windows east to sunrise and north to hills. Corner lot at main intersection but we were never bothered by noise of traffic. We had 2 vehicles and never had trouble finding parking adjacent to condo. Condo pools and BBQ were close by. Lots of green space. Grounds were in great shape. A very good place to stay.
Brenneke's beach was great for boogie boarding. Poipu beach was fabulous for laying on beach, body surfing, and wading. Snorkeling not the best but was still pretty good. Beautiful sunshine and warm blue water. Best beach on the island by far - no need to go searching for alternatives.
There is a great walk/hike along the coast going east past the Hyatt - you can go for probably 5 km's - stunning scenary and easy hike.
We took 1 day and drove around to the North end as far as road went - great day trip, highly recommended. Lots of sites to see off the beaten path
Waimea Canyon is spectacular - drive to the very end of road - worth it.
Kauai Coffee plantation worth a visit - drink as many samples as you want.
Went on a Tubing tour - tubing down old irrigation canals - worth it.
Went fishing - caught lots of tuna, had a great BBQ.
Went on catarmaran to Napali cost - stunning scenary.
We had a great trip - thanks to Jennifer for the condo - awesome.
We had no complaints at all - everything was as good or better than we expected with the condo, the location, and the amenities. We highly recommend it.
We went to Kauai with my family (2 adults, 1- one year old) and my friend's family (2 adults, 1-three year old, 1-one year old) and stayed at this condo. The accommodations were perfect. Couldn't have asked for anything more. Jennifer was a pleasure to deal with. Very responsive and her place came fully stocked with everything we could have wanted. She had two pack-and -plays (for babies to sleep in) and two high chairs, saving us from packing any baby gear with us. There were even buckets and shovels in the storage locker for the beach and snorkel gear and beach chairs for the big kids!
The kitchen and bathrooms were also fully stocked with things that other VRBOs I've been to haven't had.
Poipu is gorgeous. We were 30 seconds away from one of the pools (where a BBQ was also located) and an 8-10 minute walk down a gorgeous greenbelt to a great beach for snorkeling, sunbathing, swimming and even a protected area for the younger kids to splash around and enjoy.
This unit actually looks better live than it does in pictures. We had a fabulous time in Kauai. Poipu was amazing and this condo was perfect for us. I would recommend this to anyone and hope to return soon!
We stayed in this condo for two full weeks with 5 children under 10 and 4 adults. It was amazingly perfect. It was clean and equipped with everything we could need or want! It is a great floor plan, roomy, and all the furniture is very comfortable. I like to only stay at Hampton or Hilton Hotels and would definitely say this was just as nice or Nicer. Beds were out standing between the comfy sheet and mattresses. My husband now thinks we need a new bed. Lol. I would definitely stay there again and would recommend to any body and never second guess it! We had so much fun seeing all of the Island and found the best kids beach and boogie boarding beach just 5 min away. We had a 4 day convention at the Grand Hyatt and it was a beautiful walk over on a nice paved path. So glad we stayed at this house instead of the Hyatt. Jennifer was very patient and great to work with! We made memories that my children, in laws, husband and I will never forget. THANK YOU....
As our family of 5 has grown up we began renting condos or vacation homes and this was our best rental yet. Jennifer was great to work with. Our family was reunited, meeting our son who was living in the South Pacific. Jennifer was so flexible as I tried to arrange a complicated travel schedule with three different itineraries.
I loved the really efficient use of space in the unit. It was perfect for a family of 5. How clever to have plantation shutters to give the loft privacy. There was plenty of space to spread out and relax, as each room was appointed with its own tv.
The condo is beautifully furnished, and yet very comfortable. It was well appointed with everything that one would need, from snorkels, beach towels, to hair dryer and even spa amenities. We ate in more than half the time and was lacking for nothing in the kitchen.
While the location is the furthest end of the development, the location was was still perfect. It was a beautiful walk through a lush landscaped pathway to Poipu Beach. Or another walk in the opposite direction to Shipwrecks Beach. Both were an easy and quick drive, with available parking if you are not inclined to walk. Each of the beaches offers something different. Every day there was a variety of sea life (sea turtles or sea lions) near or in the lagoon. And there is a spectacular hike on the cliffs above Shipwreck's Beach with amazing sea views.
There are so many amenities close by to take advantage of. Be forewarned that if you ATV, it is going to be very very muddy.
Kauai is such a spectacular vacation destination that it will take several trips to truly experience everything that the island has to offer.
Jennifer is a great owner, more than willing to go out of her way to help the people who come to her condo. I wouldn't hesitate to visit again, and hope that I have the opportunity to come back soon.
Jennifer's condo is a rare gem. All furniture and fixtures are beautiful. The beds are heavenly comfortable with down duvets and pillows. My husband and I are pillow hogs, and the closets were stocked with even more pillows than we needed! Everything was beautifully clean and kept up. The master bathroom is very big, and is reminiscent of larger luxury suites in hotels. The condo is stocked with plenty of toiletries. The kitchen is stocked with more than you will need - just about anything you can think of, and Jennifer has supplied it. She has even prepared for unexpected things, such as having a notepad and pen available in the kitchen drawers etc. The dishes are all nice and stylish, not the simple white dishes and bent forks that can easily be found in other vacation rentals. The outside closet is stocked with boogie boards, snorkel gear, beach gear, plenty of towels. Literally, you just have to bring your clothes to this condo. You do not have to worry about any supplies, Jennifer has all bases covered. To top it all of, this place has the luxury feel of a hotel. I would stay here again and again and recommend it to anybody I can! The island of Kauai is an amazing place, and I am so glad I was able to stay in a great condo like this on my first trip! I WILL be back! Thank you Jennifer!
We had a great time during our stat here last month. The condo is beautiful, nicely decorated, and we loved Kauai. During our stay, the owner was super nice and even let us stay in the unit without charge a bit late to accommodate our flight times.
The condo had everything we needed on arrival other than coffee, so we left a little behind for the next guest. It's well stocked with everything you could need otherwise, including complete beach gear (although some of that stuff is not in great shape.. snorkel masks were broken, etc).
The only downside to the unit itself was that it's literally the furthest from the beach in the entire complex. With little kids and beach gear, and heat, it was a truly miserable 15-20 minute walk each way. It's really not feasible with small kids without a car, and that's a bummer in Kauai! The unit is also not a true four bedroom (one is a loft without a door that you walk through to get to another bedroom), which might be an issue.
The real issue for me was after our stay. My daughter threw up in bed the night we arrived and didn't tell anyone until morning so ALL of the linens were stained. I washed them multiple times, and her cleaning lady tried, but they were ruined. I asked how to pay for those, and she replied asking me to call her, which was weird. I have little kids so the phone is basically impossible. I didn't hear a word back until a month later when she charged me over $400 and sent a bill including chocolate on another set of bedding and a broken shower head. Neither of these were told to me beforehand, she just charged my debit card!
The problem I have is that no one even HAD chocolate during our stay. The shower head fell off when we first tried to use it so we just used another bathroom. The TV and electronics were all moved around and unhooked when we arrived, by a prior guest, and I'm pretty sure they did this stuff, too, but I just got stuck with the bill. When I mentioned (nicely) that I was surprised by these charges, she replied (not nicely) that she should've charged me for extra carpet cleaning and her cleaning lady's time trying to clean the linens, too. Holy rudeness.
I understand that this is a private home. But, my daughter puked, and now I'm out over $400, and it feels like they went over things with a fine tooth comb after that looking for anything else. As a single parent, $400 is a big deal to me.
In the future, I will stick with hotels where puke doesn't result in high fees.
Beautiful, luxurious home in paradise. This was the kind of home that you can only dream about! In fact my 18 year old nephew said that he was going to own something like this when he got older! The A/C, beds, linens, towels, kitchen everything was fabulous! The boogie boards and everything else that was supplied made our stay that much more enjoyable! The loft bedroom that is at the top of the stairs with the shutters sits right over the kitchen and dining area. This bedroom was delegated to the two people that wanted to stay up late and get up early because it's definitely not sound proof. It worked out fine with our group and there were no complaints from the two that picked that bedroom!
Jennifer was wonderful to deal with and responded immediately to any and all of my emails. We could not have asked for a nicer place and feel so fortunate to have found it on VRBO. My family did not want to move when our timeshare became available and I almost had a mutiny on my hands! We only got to stay for three days but next time we will absolutely spend more time in this beautiful home!
Thank you Jennifer! Everything was perfect! I will absolutely recommend your place to everyone I know although I don't really feeling like my sharing my good fortune!
Newly Built, Central Air-Conditioning!, Gourmet Kitchen - Granite Counters, Designer Furnishings, Spacious Bedrooms & Bathrooms, Walk-In Tile Showers and 5 Minute Walk to Poipu Beach
|Rate Period
|Weekend Night
|Monthly *
Fall Special
Sep 1 2015 - Nov 14 2015
5 night minimum stay
Seasonal Pricing
Dec 20 2015 - Jan 3 2016
7 night minimum stay
My Standard Rate
4 night minimum stay
|Tax Rate
|All major credit cards accepted, an initial payment of $1000 is required at booking, full payment is required 90 days before your arrival.
* Approximate monthly rate. Actual rate will depend on the days of the month you stay.
Payment is usually accepted in the quoted currency (USD) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.
Learn more about this vacation rental at the owner's website:


text:
716 West Addison Street (map)Sunday Worship 9:00 and 11:00 a.m.
Chicago, IL 60613
773.281.2655 Telephone
773.281.7728 Fax
773.281.7728 Fax
L.E.A.D. Conference
Joy Douglas Strome will be preaching at this year's L.E.A.D. Conference down at Fourth Presbyterian on March 7th from 8am to 3pm. Rev. John Bell of the Iona Community will be leading workshops. To register for this exciting event, click above.
Learning to Walk in the Dark
Our Lenten book study, centered on Barbara Brown Taylor's "Learning to Walk in the Dark," will commence soon, with weekly sessions at 7pm on Thursdays in Lent. Click the image above to sign up!
Daylight Savings
Reminder: spring your clocks ahead one hour this Sunday, March 8th! See you in church at 9 and 11 am!

subset: commoncrawl


text:
Palestine: 142 new Israeli settlement units East Jerusalem
Switzerland: Final day of Iran nuclear talks in Lausanne
Yemen: Saudi airstrikes kills 40 civilians in refugee camp
India: 10 killed, 30 injured as bus falls into ditch in Jharkhand
Palestine: 10 Palestinians kidnapped by Israeli forces in W Bank, Jerusalem
Palestine: Israeli navy fires on fishing boats off Gaza
Palestine: Israeli soldiers invade communities near Jenin
Australia defeat New Zealand to win Cricket World Cup
Tunisia: Amid peace march, suspected Bardo attack leader killed
Nigerians vote in tense elections marred by violence
France: Victory for France's conservatives in local elections
Indonesia: 6 killed, 4 missing in landslide
Pakistan rejects rumours of sending troops to Yemen
Palestine: Israeli settlers attack Palestinian farmers
Palestine: Shepherds detained, Israeli settlers attack farmers
Palestine: Israeli army fires missile at Gaza farmers
Syria: Idlib capital falls to al Qa'ida terrorists
Guinea declares 45-day Ebola 'health emergency'
Nigeria election extended after technical problems&violence
[File photo: Palestine TV] By Saed Bannoura IMEMC & Agencies: Wednesday night, Palestinian medical sources have reported that one Palestinian was killed, and two were seriously injured, when an Israeli war jet fired a missile targeting a fighter northwest of Gaza city. … Continued
[Still Image From Palestine TV Video] By Saed Bannoura IMEMC News: Wednesday, nonviolent Palestinian activists held a mock soccer match in front of the Ofer Prison, near the central West Bank city of Ramallah, to express solidarity with hunger striking detainees, … Continued
By Chris Carlson International Middle East Media Center: Israeli settlers stormed the park of the Ibrahimi Mosque (Cave of the Patriarchs) in Hebron, on Wednesday, where they attempted to sabotage its content, according to media sources. WAFA correspondence … Continued
Chris Carlson International Middle East Media Center: Israeli forces kidnapped three Palestinians from West Bank cities of Jenin and Hebron, Tuesday, in addition to razing Palestinian-owned lands in Jerusalem. According to Palestinian security sources, the Israeli army raided Jenin refugee … Continued
[File photo: Qudsmedia] IMEMC & Agencies: Tuesday, Israeli soldiers, heavily deployed in occupied East Jerusalem, especially near the al-Aqsa Mosque, attacked dozens of Palestinians, including children, near Bab al-Majlis Gate (The Council Gate), and prevented them from entering the mosque. Sheikh … Continued
[Photo by ISM] International Solidarity Movement: Sunday morning, Israeli police forces raided the village of Iqrit (located on the northern tip of Israel, within eyesight of Lebanon). The police uprooted trees, destroyed and confiscated all the personal belongings of Palestinian … Continued
By Saed Bannoura IMEMC & Agencies: Tuesday morning, several Israeli military jeeps and a number of armored bulldozers invaded Far'oun town, south of the northern West Bank city of Tulkarem, and demolished one home. Head of the Far'oun Town Council, … Continued
[File: Image eshahed.com] IMEMC & Agencies: Monday, Israeli soldiers, stationed across the eastern border of Gaza City, fired dozens of rounds of live ammunition targeting a number of homes, and farmlands, east of the ash-Shujaeyya neighborhood, east of Gaza city. The … Continued
By Saed Bannoura IMEMC & Agencies: Monday, Israeli soldiers kidnapped at least ten Palestinians in different parts of the occupied West Bank, and confiscated around 80 computers from two stores in Tulkarem. Local sources in Hebron, in the southern part of … Continued
By Chris Carlson International Middle East Media Center: A Palestinian fisherman who was recently shot and critically injured by the Israeli navy, off the coast of the northwest part of the Gaza Strip, has died of his wounds Sunday, … Continued
[Image By Raya News] By Saed Bannoura IMEMC & Agencies: Saturday evening, Israeli soldiers invaded Zabbouba town, near the northern West Bank city of Jenin, kidnapped an elderly man and hurled gas bombs, causing several family members to suffer the effects … Continued
[Photo: Governor of Hebron (right) Kamel Hmaid meets Governor of Palestine Monetary Authority Dr Jihad Al Wazir earlier] By Chris Carlson International Middle East Media Center: Israeli forces briefly detained the Governor of Hebron, Kamel Hmaid, in Hebron, on Saturday. … Continued
[Silwan Information Center] By Saed Bannoura IMEMC News: Friday evening, Palestinian sources have reported that one Palestinian suffered a serious head injury after Israeli soldiers opened fire at nonviolent protesters in the al-Eesawiyya town, in occupied East Jerusalem. Mohammad Abu al-Hummus, member … Continued
By Chris Carlson International Middle East Media Center: Israeli Navy boats have opened fire at Palestinian fishermen boats again, this time in the al-Sodania area, northwest of Gaza. PNN correspondence has reported that the sounds of bombs and gun shootings … Continued
(MaanImages) By Chris Carlson International Middle East Media Center: At least ten protesters were injured across the West Bank and East Jerusalem, on Friday, as dozens engaged in demonstrations marking the 47th anniversary of the Naksa, when the West Bank, Gaza … Continued
[Image: Radio Bethlehem 2000] By Saed Bannoura IMEMC & Agencies: Friday at noon, Palestinian medical sources have reported that several Palestinians suffered the effects of tear gas inhalation, and one child was injured, during clashes with Israeli soldiers in front of the … Continued
[Al Ray photo] By Chris Carlson International Middle East Media Center Editorial Group: Friday morning, 160 settlers under the protection of Israeli police marched through the courtyards of al-Aqsa Mosque from the Mughrabi Gate. Director of Media at Aqsa Foundation for … Continued
[Photo: DCI- Palestine] A total of 196 Palestinian children were imprisoned and prosecuted in the Israeli military court system in April, according to rights group Defence of Children International-Palestine (DCI-Palestine). This represents a decrease of 3 percent from March. … Continued
[Image By Hebron Defense Committee – Facebook] By Saed Bannoura IMEMC News: Thursday, Israeli soldiers attacked students holding a nonviolent protest in solidarity with the Palestinian political prisoners, in the center of Hebron city, in the southern part of the … Continued
IMEMC & Agencies: Thursday at dawn, Israeli soldiers invaded different parts of the occupied West Bank, broke into and searched several homes, and kidnapped at least five Palestinians. Hasan Breijeyya, coordinator of the Popular Committee against the Wall and settlements in … Continued


text:
Community Link Consulting (CLC) has been serving Federally Qualified Health Centers (FQHC) since 2001; providing quality, professional services that address the complex needs of over seventy clients in twenty plus states. CLC employs knowledgeable, reliable professionals to work with you to strategically develop and implement decisions that maximize the potential of your organization in all capacities, allowing you to deliver full attention to meeting the needs of your community though compassionate and effective care.
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CLC fully understands the complexities of FQHC reimbursement and revenue cycle management. Consultants can provide direct support in dashboard reporting, monthly financial statement preparation, Medicare/Medicaid cost reporting, bookkeeping services, budget development, and more.
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Sprint Nextel
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News on Sprint Nextel (Ticker: S) continually updated from thousands of sources around the net.
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text:
Ramada Montpelier Hotel and Conference Center, Montpelier
Situated in Montpelier, Ramada Montpelier Hotel and Conference Center is close to Williams County Fairgrounds. Additional regional attractions include Stoney Ridge Winery and Spangler Candy Store Museum and Tour.
Hotel Features.
Ramada Montpelier Hotel and Conference Center has an indoor pool, a spa tub, and a sauna. Complimentary wireless Internet access is available in public areas and a computer station is located on site. Dining options at this Montpelier hotel include a restaurant and a bar/lounge. Additional amenities include a fitness center, an arcade/game room, and laundry facilities. Self parking is complimentary.
The 157 air-conditioned guestrooms at Ramada Montpelier Hotel and Conference Center include coffee/tea makers and hair dryers. Guests can use the in-room complimentary wireless high-speed Internet access. Televisions are equipped with cable channels.
This was one of the worst experiences I have ever had. The bathroom had mold in the…
The back side room were not lite up in passageway, the wi fy did not work in our rooms.…
Enter dates to check prices
Hotel Info: 866-539-8117from $79
- Standard Room, 1 Queen Bed
- Standard Room, 1 King Bed
- Suite, 1 Queen Bed, Smoking
- Suite, 1 Queen Bed
- Double Room, 2 Double Beds
- Standard Room, 1 King Bed
- Deluxe Room, 1 King Bed with Sofabed
- Standard Room, 2 Queen Beds
- Double Room
- Double Room, 2 Double Beds
- Standard Room, 1 Queen Bed, Accessible
- Standard Room, 1 King Bed, Smoking
- Standard Room, 2 Queen Beds, Smoking
- Suite, 1 King Bed, Smoking
- Standard Room, 2 Queen Beds, Smoking
- Deluxe Room, 2 Queen Beds
- Suite, 1 King Bed
- Standard Room, 1 King Bed with Sofabed, Jetted Tub
Recommended hotels
At a glance
Key facts
Hotel size
- This hotel has 157 rooms
- This hotel is arranged over 3 floors
- Check-in time starts at 4 PM
- Check-out time is noon
- Express check-in
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Pets allowed
- Restrictions apply *
Extra people
- Roll-away beds available, charges may apply*
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
- Accessible parking
- Free RV/bus/truck parking
Nearby Two Star Montpelier hotels
In the hotel
- Restaurant
- Bar/lounge
- Coffee/tea in the lobby
- Indoor pool
- Fitness facilities
- Spa tub
- Sauna
- Golfing nearby
- Arcade/game room
- Number of meeting rooms - 5
- Conference space
- Meeting rooms
- Conference center
- Computer station
- 24-hour front desk
- Dry cleaning/laundry service
- Laundry facilities
- Free newspapers in lobby
- Luggage storage
- Wedding services
- Number of buildings/towers - 1
- Elevator/lift
- ATM/banking
- Safe-deposit box at front desk
- Disabled parking
- Accessible path of travel
- Braille or raised signage
- Accessibility equipment for the deaf
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- Air conditioning
- Coffee/tea maker
- Iron/ironing board
- Hair dryer
- Cable TV channels
- Free WiFi
- Connecting/adjoining rooms available
Nearby hotels
Special features
DiningWynns Restaurant - This restaurant specializes in American cuisine and serves breakfast, lunch, and dinner. Guests can enjoy drinks at the bar.
Ramada Montpelier Hotel and Conference Center, Montpelier's small print
Also known as
- Ramada Montpelier
- Montpelier Ramada
- Ramada Hotel Bryan Montpelier
- Ramada Inn Bryan Montpelier Oh Defiance
- Ramada Inn Bryan Montpelier Oh
The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.
Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation.
Optional extras
Rollaway beds are available for USD 8 per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.


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Page 1
c-Myc–Induced Extrachromosomal Elements Carry
Active Chromatin1
Greg Smith*,y, Cheryl Taylor-Kashton*,y, Len Dushnickyz, Stephen Symonsz, Jim Wright*,yand Sabine Mai*,y
*Manitoba Institute of Cell Biology, CancerCare Manitoba, the Genomic Center for Cancer Research and
Diagnosis;yUniversity of Manitoba;zCanadian Grain Commission, Winnipeg, Manitoba, Canada
Murine Pre-B lymphocytes with experimentally acti-
vated MycER show both chromosomal and extrachro-
mosomal gene amplification. In this report, we have
elucidated the size, structure, and functional compo-
nents of c-Myc–induced extrachromosomal elements
(EEs). Scanning electron microscopy revealed that EEs
isolated from MycER-activated Pre-B+ cells are an
average of 10 times larger than EEs isolated from
non-MycER–activated control Pre-B?
demonstrate that these large c-Myc–induced EEs
are associated with histone proteins, whereas EEs
of non-MycER–activated Pre B?
Immunohistochemistry and Western blot analyses
using pan-histone–specific, histone H3 phosphoryla-
tion–specific, and histone H4 acetylation–specific
antibodies indicate that a significant proportion of
EEs analyzed from MycER-activated cells harbors
transcriptionally competent and/or active chromatin.
Moreover, these large, c-Myc–induced EEs carry
genes. Whereas the total genetic make-up of these
c-Myc–induced EEs is unknown, we found that 30.2%
of them contain the dihydrofolate reductase (DHFR)
gene, whereas cyclin C (CCNC) was absent. In
addition, 50% of these c-Myc–activated Pre-B+ EEs
incorporated bromodeoxyuridine (BrdU), identifying
them as genetic structures that self-propagate. In
contrast, EEs isolated from non-Myc–activated cells
neither carry the DHFR gene nor incorporate BrdU,
suggesting that c-Myc deregulation generates a new
class of EEs.
Neoplasia (2003) 5, 110–120
cells. We
cells are not.
Keywords: c-Myc, extrachromosomal elements, gene amplification, histone modification,
genomic instability.
c-Myc is an important regulator of growth, proliferation,
differentiation, and development. Deregulation of the c-Myc
oncoprotein has been reported in apoptosis (for review,
see Ref. [2]), cellular transformation (for reviews, see, Refs.
[3–5]), and in malignancies of lymphoid and nonlymphoid
origin [6–9]. c-Myc is involved in repressing, enhancing, and
initiating transcription [5–11]. Recent evidence links c-Myc
to angiogenesis [12–14].
The deregulation of c-Myc is a common feature in many
tumors, where it is frequently found translocated and/or
amplified [15,16]. c-Myc promotes replication [17] and
genomic instability [18–25]. This study addresses the
mechanismsof c-Myc–dependent
Genomic instability refers to genetic changes that affect
thenormalorganization and
chromosomes. These alterations may be structural or
numerical. Factors such as genotoxic stimuli, growth
factors, and oncogenic deregulation are potent inducers of
genomic instability (for reviews, see, Refs. [26–28]). It
was found previously that the deregulation of c-Myc
expression under both in vitro and in vivo conditions leads
to karyotypic (chromosomal) and locus-specific genomic
instability [18–25]. Earlier, we showed c-Myc–dependent
amplification of the dihydrofolate reductase (DHFR) gene
for hamster and rat fibroblasts, as well as in human breast
cancer and mouse plasmacytoma cell lines [20]. DHFR
was also amplified in vivo during the first week of c-Myc–
dependent induced plasmacytomagenesis in BALB/c mice
[29], as well as in fully developed primary mouse
plasmacytomas [30]. More recently, we have described
the c-Myc–dependent generation of extrachromosomal
elements (EEs) in a mouse Pre-B lymphocyte cell line
(designated Pre-B) [22,31,32]. To date, this mouse cell
line has been shown to carry the following amplified genes
extrachromosomally and intrachromosomally upon c-Myc
deregulation: DHFR [31],
ribonucleotide reductase R2 (R2) [22], and ornithine
decarboxylase (ODC) [33].
function ofgenes and
cyclin D2(CCND2)[21],
Neoplasia . Vol. 5, No. 2, March/April 2003, pp. 110–120
Abbreviations: BSA, bovine serum albumin; BrdU, bromodeoxyuridine; CCND2, cyclin D2;
CCNC, cyclin C; DAPI, 40,60-diamidino-2-phenylindole; DIG, digoxygenin; DHFR,
dihydrofolate reductase; EEs, extrachromosomal elements; FISH, fluorescent in situ
hybridization; FISH-EEs, fluorescent in situ hybridization of extrachromosomal elements;
4-HT, 4-hydroxytamoxifen; fIHC, fluorescent immunohistochemistry; GAPDH, glyceralde-
hyde-3-phosphate dehydrogenase; H3P, phosphorylated histone H3; ICD, interchromo-
somal domain; kDa, kilodalton; MycER, Myc estrogen receptor construct (1); ODC,
ornithine decarboxylase; Pre-B? cells, non–4-HT–activated murine Pre-B lymphocyte
cells; Pre-B+ cells, 4-HT–activated murine Pre-B lymphocyte cells; Pre-B (abl), murine
Pre-B lymphocyte cells that do not harbor the MycER construct but only the Abelson virus;
R2, small subunit of ribonucleotide reductase; SDS-PAGE, sodium dodecyl sulphate
polyacrylamide gel electrophoresis; SEM, scanning electron microscopy; spcDNA, small
polydispersed DNA; Txred, Texas Red fluorochrome
Address all correspondence to: Sabine Mai, Manitoba Institute of Cell Biology, 675
1Funding for this research was provided by the Canadian Institutes of Health Research and
by the National Sciences and Engineering Research Council of Canada.
Received 17 July 2002; Revised 28 November 2002; Accepted 2 December 2002.
Copyright# 2003 Neoplasia Press, Inc. All rights reserved 1522-8002/03/$25.00
Page 2
EEs have been found in all cells, including primary cells
[34]. For immortalized human fibroblasts, EEs range in
number from 10 to 60 molecules per cell and average 0.8 ?m
in diameter [34]. These smaller circular molecules are also
called small polydispersed DNA (spcDNA) and carry
repetitive DNA sequences [35,36]. In contrast, large
(averaging 1–2 ?m in diameter) chromatin structures, called
episomes and double minutes, which are included in the
broad category of EEs [34], have long been known to harbor
amplified genes (for review, see Refs. [37–40]). This class
of EEs is larger than 250 kb in size and can number in the
thousands, depending on which tumor cell line or tumor is
analyzed. In 1979, Kaufman et al. [41] first showed the
generation of EEs carrying amplified DHFR in unstable
methotrexate-resistant mouse cell lines. More recent stud-
ies on EEs have shown that these circular double-stranded
DNA molecules are prevalent in cancer cells and harbor
oncogenes, in addition to drug resistance genes [42–44].
There have been very few studies addressing the
mechanism of EE formation and whether or not these
molecules function as ''active chromatin units.'' Studies by
Itoh and Shimizu [45] showed that EEs localize to the
nuclear interior upon entry of the cell into S-phase.
Interestingly, Solovei et al. [46] showed that EEs found in
the neuroblastoma cell line HD-N-16 associate with the
interchromosomal domain (ICD). It was suggested that this
ICD harbors macromolecular complexes that are involved in
transcription, splicing, DNA replication, and repair [47,48].
However, compelling data on these cellular functions and
their association with EEs have been lacking.
Here, we have analyzed for the first time the size,
structure, and functional composition of c-Myc–induced
EEs. We report that EEs generated in MycER-activated [1]
Pre-B cells carry competent and/or active chromatin. In
addition, we show that only c-Myc–induced EEs harbor
genes such as DHFR. Moreover, we describe that these c-
Myc–induced EEs have the potential to replicate, whereas
EEs isolated from non–c-Myc–activated Pre-B cells do not
have this ability. The genetic composition of c-Myc–induced
EEs is nonrandom; DHFR is present on EEs whereas cyclin
C is not. Importantly, EEs from non–c-Myc–induced control
cells do not contain these genes.
Materials and Methods
Cell Lines
Pre-B mouse lymphocyte line (designated Pre-B)
[21,22] and Pre-B (abl) cells [49] were cultured in RPMI
1640 medium supplemented with 10% (vol/vol) fetal bovine
serum, 1% (vol/vol) sodium pyruvate, 50 IU/ml penicillin,
50 ?g/ml streptomycin, 5.5?10?5M b-mercaptoethanol,
and 2 mM glutamine (Canadian Life Technologies, Burling-
ton, Ontario, Canada). The Pre-B cells were seeded for
24 hours, followed by c-Myc activation for 72 hours using
100 ?M 4-hydroxytamoxifen (4-HT) (Sigma, Oakville,
Ontario, Canada) dissolved in 100% ethanol. The 4-HT–
activated cells were designated Pre-B+ and the non-
activated Pre-B cells were designated Pre-B?, which in
MycER activation experiments received an equal volume of
100% ethanol. MycER activation was determined as
described earlier, using quantitative fluorescent immunohis-
tochemistry (fIHC) techniques [22].
fIHC of Purified EEs
fIHC was performed as described previously on Pre-
B+ and Pre-B? EEs [31], which were isolated using the
Hirt procedure [50]. The purity of the EE preparations
was examined using the fluorochrome 40,60-diamidino-2-
phenylindole (DAPI), which specifically intercalates into
AT-rich regions of DNA. Subsequent analysis of EEs from
Hirt extracts by fluorescent microscopy identified whether
ornot therewasgenomic
electron microscopy (SEM) was used to confirm these
staining results. As a result, EE preparations containing
DNA fibers, as detected by either of the two methods,
were excluded from our analysis [31]. Only purified
EEs devoid of contamination by genomic or apoptotic
DNA were used in all of the experimental procedures
listed below.
Where indicated, cross-linking of EEs and protein was
carried out for 10 minutes at room temperature with 1%
formaldehyde in 1? phosphate-buffered saline/50 mM
MgCl2. Sheep anti-core histone antibody (United States
Biological, SwampScott, MA) was used at 5 ng per slide
and visualized with a FITC-conjugated donkey anti–sheep
IgG antibody (Sigma) at 2.75 ng per slide. The anti–
histone H3 phosphorylation–specific antibody used is a
histone H3 phosphoserine monoclonal antibody from Juan
et al. [51]. It was used at 4.0 ng per slide and visualized
with a Texas Red conjugated goat anti–mouse IgG1
antibody (Southern Biotechnology Associates, Birmingham,
AL) at 2.5 ng per slide. Image acquisition was performed
using a Zeiss Axioplan2 microscope (Carl Zeiss Canada,
Toronto, Ontario, Canada) under a 63? oil immersion
objective and a UV filter. The images were assessed using
Northern Eclipse 5.0 software (Empix Imaging, Missis-
sauga, Ontario, Canada) and a Hamamatsu CCD camera
(East Syracuse, NY).
Bromodeoxyuridine (BrdU) Replication and Gene
Expression Analysis of EEs
A total of 2.0?106Pre-B cells was stimulated with
100 ?M 4-HT or an equal volume of 100% ethanol and
grown for 72 hours, followed by a BrdU pulse (Sigma) at
a concentration of 10 ?M for 30 minutes before harvest-
ing. The EEs were then isolated using the Hirt extraction
purified EEs were subjected to fluorescent in situ hybrid-
ization (FISH) as described previously [31], using the
following probes: dihydrofolate reductase (DHFR) [20]
and cyclin C [20] conjugated to either digoxigenin (DIG)
or biotin, respectively. The DIG-labeled probes were
detected using a 1:200 dilution of anti–DIG-FITC anti-
body(Roche Diagnostics,
previously[31,53]. The
Laval, Quebec,Canada),
Neoplasia . Vol. 5, No. 2, 2003
c-Myc–Induced Extrachromosomal Elements
Smith et al.
Page 3
whereas the biotin-labeled probes were detected using a
1:200 dilution of monoclonal mouse anti–biotin antibody
(Roche Diagnostics), followed by a 1:400 dilution of goat
anti–mouse IgG Texas Red secondary antibody (South-
ern Biotechnology,Associates,
parallel, BrdU that was incorporated into the EEs was
detected as reported previously [32]. Analyses were
performed using a Zeiss Axiophot microscope (Carl Zeiss
Canada) under a 63? oil immersion objective and UV/
FITC and Texas Red filters coupled to a Photometrics
CH250/A CCD camera (Eastman Kodak, Rochester, NY).
The images were assessed using the IP Lab version 3.1
software (Scanalytics, Fairfax, VA).
Birmingham, AL).In
Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) and Silver Staining
of SDS-PAGE Gels
Silver staining of 15% SDS-PAGE gels was performed
as outlined by the manufacturer of the silver stain oxidizer,
silver reagent, and developer solutions (Bio-Rad Labora-
tories, Hercules, CA). Fifteen percent SDS-PAGE gels
were made, loaded, and electrophoresed as outlined by
Laemmli [52].
Protein gels for silver stain and Western blot analysis
were loaded with 2 ?g of low molecular weight standard;
either 5 or 10 ?g of total histone extract from chicken
erythrocytes; 40 ?g of ovalbumin and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH); 20 ?g of bovine
serum albumin (BSA) and anti–mouse whole IgG anti-
body; either 25 ?l, 50 ?l, 40 ?g, or 0.5 ?g (based on
DNA concentration) of Pre-B+ / ?
(basedon DNA concentration)
splenocyte EEs.
EEs; and 0.5 ?g
of C57BL6mouse
Western Blot Analysis of EEs
Formaldehyde fixed or nonfixed Pre-B? / + and Pre-B
(abl)? / + EEs as well as EEs isolated from primary
C57BL6 mouse splenocytes were isolated using the Hirt
procedure [50] and subjected to three rounds of dialysis
using ddH2O as the exchange buffer. Dialysis tubing
(Canadian Life Technologies) (3/4 in. diameter; 12,000
kDa molecular weight exclusion limit) was used to dialyze
a large volume of EEs, followed by their concentration
using a Microcon 50 (50,000 kDa cutoff) concentrator
(Amicon, Bedford, MA). After centrifuging repeatedly at
13,000rpm,theEEs were
contamination as described above. The concentration of
the protein containing EEs was then determined by using
either a standard Bradford protein assay [54] using BSA
concentration was determined by using a fluorometric
assay. In this assay, the DNA concentration of the EEs
was determined by resuspending a purified EEs aliquot in
a buffer containing 25 mM sodium phosphate buffer
(monobasic) and 0.01 M DAPI, pH 7.5. The plasmid
pcDNA3 (Invitrogen, Carlsbad, CA) was used as the
concentration standard.The
standard,or theDNA
concentration wasthen
determined by using the Titerek Fluoroskan II fluorometer
(Labsystems, McLean, VA) at an absorbance/emission
spectra specific to DAPI. The measured fluorescence was
then analyzed using Spectrosoft software (Labsystems).
Depending on the Western blot experiment performed,
either a 40-?g quantity of protein containing EEs (as
determined by the Bradford assay) or 0.5 ?g of EEs (as
determined by the fluorometric assay) was then loaded on
a 15% SDS-PAGE gel, along with total histone isolated
from chicken erythrocytes. Negative controls such as
ovalbumin (Sigma ImmunoChemicals, Oakville, Ontario,
Canada), GAPDH (Sigma), BSA (Sigma), and anti–
mouse IgG (Sigma) were loaded. After electrophoresis of
the protein samples for 2 hours at 100 V, protein was
transferred to a Hybond-C Super membrane (Amersham
Pharmacia Biotechnologies, Piscataway, NJ) using a Bio-
Rad (Mississauga, Ontario, Canada) semidry transfer
apparatus Transblot SD. To optimize the transfer of highly
basic proteins such as histones, a 3-[cyclohexylamino]-
1-propanesulfonicacid (CAPS)
utilized (25 mM CAPS, pH 10, 20% methanol), allowing
overnight transfer at 50 V (Bio-Rad). Blocking was then
performed the next day for 15 minutes using 3% skim milk
powder (wt/vol) in 1?/0.2% Tween-20 buffer. For detec-
tion of acetylation-specific species of H4 histone, a 1:1000
unit dilution of rabbit penta H4-hyperacetylated specific
antibody (Upstate Biotechnology, Hornby, Ontario, Can-
ada) was used for 1-hour incubation under rocking
conditions, followed by three 5-minute washes in 1?
TBS/0.2% Tween-20 solution. This was followed by
detection using a rabbit-specific IgG antibody conjugated
to horseradish peroxidase (concentration/time specified by
the company; ECL Kit, Amersham Pharmacia Biotechnol-
ogies). Visualization of the Western blot was done using
Hyperfilm ECL high-performance chemiluminescence film.
The film was then developed using the CP1000 developer
(AGFA, Etobicoke, Ontario, Canada).
transfer bufferwas
SEM of EEs
The procedure for SEM of EEs was done as outlined
previously [55,56], except for the following changes.
Suspensions of Hirt-isolated EEs mixed with an equal
volume of fresh methanol/acetic acid (3:1 vol/vol) were
dropped on cooled cover slips and then permitted to dry. As
indicated, some EE samples were treated for 24 hours at
568C with 100 ?g (10 ?g/?l) of proteinase K before SEM
analysis. The coverslip was then plunged into 50% acetic
acid solution for 1 second followed by subsequent 1-
second incubations in coplin jars filled with a mixture of
ethanol:distilled water:methanol:acetic acid (20:20:9:1 parts
by volume), 70%, 90%, and 100% ethanol solutions,
respectively. Coverslips were then cut with a diamond
pencil and mounted on stubs using liquid carbon. The
samples were then coated with 15 nm each of Gold in a
Hummer VII (Anatech, Alexandria, VA) sputter coater and
analysed at 10 kV with a JEOL JSM-6400 scanning
electron microscope (Peabody, MA) and photographed on
Kodak TMAX black-and-white film.
c-Myc–Induced Extrachromosomal Elements
Smith et al.
Neoplasia . Vol. 5, No. 2, 2003
Page 4
Ultrastructural Analysis of EEs Isolated from MycER-
Activated and Control Pre-B Cells
SEM was employed to examine the ultrastructure of
c-Myc–induced EEs. A direct comparison of EEs isolated
from non–c-Myc deregulated cells (Pre-B? ) and of EEs
isolated from c-Myc deregulated cells (Pre-B+ ) was
performed. The Pre-B? EEs were 0.1–0.2 ?m in diameter
(Figure 1, A and B), whereas c-Myc–activated Pre-B+ EEs
ranged in size from 1.0 to 20 ?m (Figure 1C), with 90% of
Pre-B+ EEs being approximately 1.0 ?m in diameter. Thus,
the EEs isolated from MycER-activated cells were an
average of 10-fold larger in diameter than Pre-B? EEs
isolated from nonactivated cells, whereas some Pre-B+
EEs were up to 200-fold larger.
focused on their protein content, their transcriptional activity,
and genetic composition, as well as on their potential
to replicate.
of c-Myc–inducedEEs
Identification of Histone Proteins on c-Myc–Induced
Pre-B+ EEs
The protein-like ''globular'' structures on Pre-B+ EEs
were not usually apparent on the Pre-B? EEs (Figure 1,
A–C). Previous reports have shown that smaller EEs found
in primary cells do not contain proteins on their surface [34].
However, because previous SEM studies have acknowl-
edged similar structures on EEs isolated from tumorigenic
cells as possibly being composed of proteins [55,56], we
wanted to determine the nature of the ''globular'' structures
found on c-Myc–induced Pre-B+ EEs. Therefore, we
pretreated samples of Pre-B+ EEs with proteinase K before
SEM analysis. Subsequent analysis showed that the overall
structure of the Pre-B+ EEs disintegrated as a result of
proteinase K treatment, suggesting that Pre-B+ EEs carry
proteins (data not shown).
Histone proteins constitute one of the major components
of nuclear chromatin material (for review, see Ref. [57]).
Many EEs can replicate autonomously [38–40] and
coordinately move with mitotic chromosomes [58], such
that they can become accessible to the cellular replication
and transcription machinery. Therefore, it is very likely that
some of the protein components of EEs are core histone
proteins (i.e., H2A, H2B, H3, and H4) (for review, see
Ref. [57]). To date, there has been no identification of the
protein components found on EEs, except for the identi-
fication of lamin B found on EEs that were isolated from
COLO 320DM cells [59].
To examine the potential presence of histone proteins
on c-Myc–induced EEs (Pre-B+ EEs), we performed
fIHC on purified Pre-B+
antibody, which detects the core histone proteins, we
identified that 25.0±3.3% of Pre-B+ EEs (Figure 2, A–D,
see arrows) contain core histone protein (Figure 2B, green
signals, see arrows, and Table 1). To determine whether
the Pre-B+ EEs contained active chromatin, we performed
fIHC using an anti–histone H3 phosphorylation (H3P)–
specific antibody. This antibody detects a modification of
histone H3 that is associated with transcriptionally active
chromatin [51]. Our data show that 20.0±3.2% of Pre-B+
EEs contained H3P protein (Figure 2C, red signals, see
arrows, and Table 1). Eighty percent of the H3P signal
overlayed with the core histone signal (Figure 2D, see
arrows). When EEs in non–c-Myc–activated control cells
EEs. Using a pan-histone
Figure 1. SEM of Pre-B? / + EEs. Representative examples of EEs isolated
from Pre-B? or Pre-B+ cells (A, B, and C, respectively). Arrows in (A) and
(B) point to small protein-free EEs (see alsoRef. [34]). Arrows in (C) point to
examples of ''globular'' structures found on the surface of the c-Myc–induced
Pre-B+ EEs. The magnification of the structures shown in (A)–(C) is
30,000?, 30,000?, and 3500?, respectively. The scale bar found at the
bottom of (A) and (B) represents 1 ?m, while the scale bar on the bottom of
panel C represents 10 ?m.
Neoplasia . Vol. 5, No. 2, 2003
c-Myc–Induced Extrachromosomal Elements
Smith et al.
Page 5
9.3±2.4% of the EEs contain core histone (Table 1). In
addition, only 9.0±1.5% of Pre-B? EEs analyzed showed
a H3P-specific signal (Table 1). These data suggest that
the majority of c-Myc–induced EEs carry transcriptionally
competent and/or active chromatin.
To eliminate the possibility that 4-HT, used to activate the
Myc-ER constructin the Pre-B cells (Materials and Methods
section), caused the generation of transcription-associated
histones on these EEs, we performed fIHC on EEs isolated
from Pre-B cells that did not harbor the MycER construct.
The cells examined, designated Pre-B (abl), contained,
similar to the Pre-B cells studied here [21], the Abelson
leukemia virus [49]. A total of 11.6±5.2% of the EEs isolated
from non–4-HT–treated Pre-B (abl) cells contained core
histone protein and 10.7±2.8% of these EEs show H3P
specific signal (Table 1).Similarly, 7.5±2.3% ofEEs isolated
from 4-HT–treated Pre-B (abl) cells contained core histone
protein and 6.8±2.8% of these EEs showed a H3P-specific
signal (Table 1). In conclusion, histone proteins were found
at low frequencies on Pre-B? EEs and Pre-B (abl)? / +
EEs irrespective of the treatment with 4-HT. In contrast, a
significant amount of c-Myc–induced Pre-B+ EEs carried
EEs) were analyzed, we found that only
histones. Thus, 4-HT does not contribute towards the
generation of EEs, but rather the deregulation of c-Myc
induces the generation of EEs with immunohistochemical
evidence of active chromatin.
Figure 2. Immunohistochemistry of EEs isolated from nonformaldehyde-treated Pre-B+ cells using both core histone and H3 phosphorylation–specific antibodies.
(A) DAPI-stained Pre-B+ EEs. (B) Pre-B+ EEs that were detected with a fluorescein (FITC)-conjugated core histone antibody. (C) Pre-B+ EEs that were
detected with a Texas Red (TR)–conjugated H3 phosphorylation–specific antibody. (D) Overlay of panels A–C. The arrows shown in each panel depict
representative examples of EEs that are highlighted by either DAPI or histone antibodies. Due to the overlay in (D), each dot appears white in color. This represents
a colocalization of signal from panels A to C. Inserts within the individual pictures highlight the respective EEs. For quantification of these findings, see Table 1.
Table 1. Core Histone and H3-Phosphorylated Protein Found on EEs.
Sample of EEs 4-HT
of cells
of EEs
Percent of
EEs with
core histone
Percent of
EEs with
H3P histone
Pre-B (abl)
This table outlines all the different EE preparations that were used for these
studies along with their corresponding core histone and H3 phosphorylation
protein levels as measured by fIHC. The percentages and standard errors
calculated are based on a total number of 300 EEs that were counted for
each sample. The analysis of 100 EEs in each of three individual experiments
was performed.
c-Myc–Induced Extrachromosomal Elements
Smith et al.
Neoplasia . Vol. 5, No. 2, 2003
Page 6
Effects of Formaldehyde Cross-Linking of Pre-B Cells on
the Amount of Histone-Carrying EEs Recovered During Hirt
Extraction of EEs
To determine the relative proportions of core histone
protein and H3P that remain associated with EEs after Hirt
isolation [50], we cross-linked EEs and protein prior to the
Hirt isolation procedure using formaldehyde (Materials and
Methods section). Formaldehyde leads to cross-linking of
proteins to DNA [60]. To this end, EE isolation and fIHC
analysis were carried out following cross-linking with 1%
formaldehyde. As a result, 71.0±10.6% of the Pre-B+ EEs
analyzed contained core histone protein (Figure 3B, see
arrows, and Table 1). In addition, 62.0±4.2% of the Pre-B+
EEs contained H3P-specific protein (Figure 3C, see arrows,
and Table 1) and the H3P signals overlayed with 85% of the
core histone signals (Figure 3D, see arrows). In contrast,
only 28.7±2.0% of Pre-B? EEs that were isolated from
formaldehyde-pretreated Pre-B cells contained core his-
tone protein (Table 1) and 28.0±1.3% of these EEs
contained H3P-specific protein (Table 1). Interestingly,
Pre-B (abl) EEs isolated from formaldehyde cross-linked
4-HT-treated cells gave similar results to Pre-B? EEs,
whereby 25.3±2.6% of EEs contained core histone protein
and 9.0±2.7% EEs contained H3P-specific protein. These
data suggest that the formaldehyde cross-linking procedure
increases the amount of histone protein bound to EEs,
providing evidence that the Hirt isolation procedure caused a
loss in the number of histone-bound EEs. In addition, using
this experimental approach, we have shown that the histone
protein content of EEs isolated from either formaldehyde-
treated Pre-B? cells or Pre-B (abl) cells is not affected by
pretreating these cells with 4-HT. Therefore, c-Myc activa-
tion specifically affects the number of core histone and H3P
histone proteins found on EEs isolated from Pre-B+ cells.
Further Molecular Analyses of Histone Proteins in c-Myc–
Induced Pre-B+ EEs
Because the fIHC data suggest that the Pre-B+ EEs
contain core histone proteins, we set out to analyze the
histone protein content further using Western blot analysis.
Because formaldehyde pretreatment of cells increased the
amount of core histone protein that was identified by fIHC, all
further analyses were performed using EEs isolated from
Pre-B cells that had been pretreated with formaldehyde.
Silver stain analysis of four independent samples of Pre-
B+ EEs revealed the presence of major protein complexes
Figure 3. Immunohistochemistry of EEs isolated from formaldehyde-treated Pre-B+ cells using both core histone and H3 phosphorylation–specific antibodies.
The fIHC experiment was performed and analyzed as outlined for Figure 2. (A)–(D) represent similar pictures as in Figure 2, except the EEs used in this experiment
were isolated from formaldehyde-treated Pre-B+ cells. Inserts within the individual pictures highlight the respective EEs. For quantification of these findings, see
Table 1.
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c-Myc–Induced Extrachromosomal Elements
Smith et al.
Page 7
at approximately 14 kDa, which were absent in four
corresponding samples of Pre-B?EEs (Figure 4A, lanes
1–4 and 5–8, respectively, see arrow). This major protein
complex migrated at the same molecular weight (14 kDa) as
total histone extract isolated from chicken erythrocytes
(Figure 4A, lane 9). Silver stain analysis of Pre-B? and
Pre-B+ EEs samples also identified many less abundant
proteins ranging in molecular weight from less than 14 kDa to
greater than 66 kDa.
We chose to study the 14-kDa proteins on EEs due to
their predominance in Pre-B+ cells and their absence in
Pre-B? cells. An antibody specific to the modified H4-
acetylated histone protein was used for this analysis
because it was previously shown that this H4 histone
modification is associated with transcriptionally active
chromatin [60]. We did not use the histone H3P antibody
because it does not recognize histone H3P protein in
Western blot analyses. We found that H4-acetylated histone
protein was present in the Pre-B+ EE sample (Figure 4B,
lane 6), whereas no H4-acetylated protein was detected in
Pre-B? EEs (Figure 4B, lane 7). The specificity of the H4
acetylation–specific antibody was confirmed by identifying
the IgG heavy chains (50 kDa) and the IgG light chains (25
kDa) of an anti–mouse IgG antibody that was used to
reprobe the original blot (Figure 4C).
We next performed Western analyses using EE samples
based on their DNA content (Figure 4D). Under these
experimental conditions, we observed a faint band for Pre-
B? EEs, indicating that Pre-B? EEs contain some H4-
acetylated protein (Figure 4D, lane 5). A stronger band was
detected for Pre-B+ EEs (Figure 4D, lane 6). To investigate
whether the immortalization process of the Pre-B cells might
be the reason why some H4-acetylated protein was found in
the Pre-B samples [34], we examined EEs isolated from
primary mouse C57BL6 splenocytes. No measurable
amount of histone H4-acetylated protein was detected in
this sample (Figure 4D, lane 7). To control for loading, the
gel in Figure 4D was silver-stained (Materials and Methods
section) (Figure 4E). Equal loading was observed for Pre-
B?, Pre-B+, and C57BL6 EEs, respectively (Figure 4E,
lanes 5–7, respectively). We conclude that c-Myc–induced
Pre-B+ EEs contain significantly more H4-acetylated
protein than Pre-B? EEs.
c-Myc–Induced EEs Carry Genes and Can Replicate
To prove that not only Pre-B+ EEs contain transcrip-
tionally competent and/or active DNA but also genes, we
performed FISH of EEs (FISH-EEs) [31] on both Pre-B?
and Pre-B+ EEs. To assess their potential to replicate, we
pulse-labeled the cells with BrdU prior to the isolation of
Figure 4. Silver stain and Western analysis of Pre-B? / + and primary spleen cell EEs using H4 acetylation–specific antibody. (A) Fifteen percent silver-stained
SDS-PAGE gel. Pre-B+ / ? EEs (lanes 1–4 and 5–8, respectively), histone extract (lane 9), and low molecular weight standard were loaded as outlined in the
Materials and Methods section. (B) Western blot using an anti–rabbit H4 acetylation–specific antibody. Low molecular weight standard, histone extract, ovalbumin,
GAPDH, anti–mouse whole IgG antibody (lanes 1–5, respectively), and Pre-B+ / ? EEs (lanes 6 and 7, respectively) were loaded as outlined in the Materials and
Methods section. (C) Western blot of the same membrane used in (B), but it was stripped and reprobed with an anti–mouse whole IgG antibody. (D) Western blot
using H4 acetylation–specific antibody. Histone extract and BSA (lanes 1 and 2, respectively), no sample (lanes 3 and 4), Pre-B? EEs, Pre-B+ EEs, and
C57BL6 mouse spleen EEs (lanes 5–7, respectively) were loaded as outlined in the Materials and Methods section. (E) Silver-stained 15% SDS-PAGE gel of the
Western blot shown in panel D.
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Smith et al.
Neoplasia . Vol. 5, No. 2, 2003
Page 8
EEs (Materials and Methods section). The simultaneous
analysis of BrdU incorporation signals and DHFR FISH
signals allowed us to conclude that: 1) Pre-B+ EEs, but
not Pre-B? EEs, incorporated BrdU; 2) Pre-B+ EEs that
contained BrdU also contained the DHFR gene, but not the
cyclin C control gene; and 3) in contrast, Pre-B? EEs did
not incorporate BrdU, and they did not contain the DHFR
gene (Table 2). We conclude that c-Myc activation in
mouse Pre-B cells leads to the generation of EEs that
contain DHFR, which has the ability to replicate. It is likely
that other genes are also present on these EEs because
previous studies have identified genes such as R2 [22]
and cyclin D2 (CCND2) [21] as being found amplified on
Pre-B+ EEs. However, only the DHFR gene was analyzed
in this study.
In 1990, Gaubatz [34] reported that all cells, including
primary cells, have a heterogeneous population of EEs
carrying repetitive DNA sequence motifs. Extrachromoso-
mal DNA is a common form of DNA amplification that is
seen in a variety of different tumors [61]. For example, in
acute myelogenous leukemia, myelodysplastic syndrome,
and acute lymphoblastic leukemia, generation of EEs is
associated with disease progression [62,63]. The signifi-
cance of EEs in tumorigenicity was shown by studies
outlining the elimination of double minute–encoded c-myc
genes from the colon cancer cell line COLO 320DM, which
reduced the tumorigenicity in mice [64]. Recently, a group
of patients with advanced ovarian cancer was treated with a
noncytotoxic dose of hydroxyurea. These patients had a
45% to 60% reduction in the number of EEs, and two
patients had a stabilized disease for 6 and 10 months,
respectively [65]. The role of extrachromosomal c-myc in
tumorigenesis was also documented in a translocation-
negative mouse plasmacytoma: c-myc was transcribed
from c-myc/IgH–containing EEs, suggesting that the
functional extrachromosomal juxtaposition of c-myc and
IgH replaced the need for a chromosomal c-myc/IgH
translocation [66].
Prior to this study, the size, ultrastructure, and protein
composition of c-Myc–induced EEs were unknown. Here,
we have shown that c-Myc–induced EEs are 10- to 200-
fold larger than EEs found in non–MycER-activated Pre-B
cells (Figure 1). To date, only two ultrastructural SEM
studies on EEs have been performed: one using EEs
isolated from COLO 320DM cells and another using EEs
isolated from epidermoid KB-V1 carcinoma cells [55,56].
The EEs isolated from these two tumor cell lines have been
well characterized, in which the c-myc and multidrug
resistance genes have been identified, respectively. The
extrachromosomal DNA analyzed from COLO 320DM cells
and KB-V1 cells was 1 and 0.5 ?m in diameter, respectively.
These results are similar to our own SEM analysis shown
in Figure 1, in which 90% of our Pre-B+ EEs isolated were
1 ?m in diameter.
We show for the first time that c-Myc deregulation leads
to the de novo generation of EEs. These EEs are larger than
the ones found in non–c-Myc–activated cells [34]. Thus, c-
Myc deregulation can also promote genomic instability by
generating a pool of extrachromosomal DNA substrates for
recombination. Moreover, this study has shown that many of
these EEs carry histones and genes and have the ability to
We demonstrated that the percentage of c-Myc–
induced EEs in Pre-B+ cells that carried transcriptionally
active histones was significantly higher than the percentage
found in Pre-B? cells and Pre-B (abl) cells (62.0±4.2%,
28.0±1.3%, and 9.0±2.7%, respectively). However, not all
of the Pre-B+ EEs have histone proteins on their surface.
This finding illustrates the heterogeneity of EEs isolated
from a particular cell type [34]. Western blot analysis further
confirmed the potential transcriptional capability of Pre-B+
EEs (Figure 4). These data are similar to studies using the
Rat1 fibroblast cell line containing the same MycER
construct as the one found in the Pre-B cell line, in which
c-Myc activation occurs before H4 acetylation [67]. In
addition, it has been shown that the activation of c-Myc in
mouse fibroblast 3T3 cells leads to the acetylation of
histones H3 and H4 at the cyclin D2 promoter [68].
Therefore, it seems likely that c-Myc is directly involved in
preparing the DNA for transcription through the acetylation
of histone proteins not only on the chromosomes, but also
on EEs.
The Western blot analyses data in Figure 4D indicate that
atleast some ofthe non–c-Myc–activated Pre-B? EEsare
transcriptionally competent and/or active due to a detectable
level of acetylated histone H4 protein. Because the Pre-B
cell line is immortalized, we examined EEs directly isolated
from mouse C57BL6 primary spleen cells. We did not identify
any acetylated histone H4 protein in EEs from these primary
Table 2. BrdU Incorporation and Detection of Genes on EEs.
Samples of EEs
Experiment 1Experiment 2Experiment 3
Percent EEs
incorporating BrdU
Percent EEs
incorporating BrdU
Percent EEs
containing DHFR
Percent EEs
incorporating BrdU
Percent EEs
containing Cyclin C
This table outlines the percentage of Pre-B? and Pre-B+ EEs that were detected for BrdU incorporation as well as the genes DHFR and cyclin C (as determined
by FISH-EEs) [31]. The percentages and standard errors calculated are based on a total number of 110 EEs that were counted for each sample in each
specific experiment.
Neoplasia . Vol. 5, No. 2, 2003
c-Myc–Induced Extrachromosomal Elements
Smith et al.
Page 9
C57BL6 cells (Figure 4D, lane 7). This result suggests that
cellular immortalization may promote the formation of EEs
(see also Ref. [34]), some of which carry H4-acetylated
protein as was detected on Pre-B? EEs.
If c-Myc–activated Pre-B+ EEs contain transcriptionally
competent and/or active DNA, then it is likely that they
contain active genes and that those may be able to replicate.
Although not all genes have been identified in Pre-B+ EEs,
those identified to date are important for DNA synthesis and
cell cycle progression. In fact, we have previously shown that
Pre-B+ EEs contain genes such as R2 [22], cyclin D2 [21],
and DHFR [20], whereas Pre-B? EEs do not carry these
genes. In addition, cell lines such as human breast ductal
carcinoma T47D, mouse plasmacytoma MOPC 265, and
MOPC 460D and human colorectal carcinoma line 320 HSR
have all shown c-Myc–dependent generation of DHFR-
containing EEs [20]. The present results suggest a possible
correlation between histone-containing EEs and gene-
containing EEs. We recently confirmed this correlation by
using anti–core histone antibody to isolate core histone–
containing EEs, followed by FISH analysis [53]. In the
present study, we have identified Myc-induced EEs that
carry histones and genes and are able to replicate (Table 2).
Based on our current knowledge, we propose a model of
c-Myc–dependent genomic instability (Figure 5), in which
c-Myc deregulation can lead not only to genomic instability
at the chromosomal level, but also at the extrachromosomal
level, with the potential for further genomic instability and
neoplasia. The novelty of the present study lies in the fact
that the structure of c-Myc–induced EEs is comparable to
the structure of chromosomes, and that c-Myc–induced
EEs appear to be functional genetic units, which are able to
transcribe and replicate the genes they carry and thus able to
contribute to cellular transformation. Therefore, by targeting
transcriptionally active genes that are found on EEs, it might
be possible to reduce or even eliminate the tumorigenic
potential of transformed cells in which c-Myc deregulation
plays a major role.
We extend our thanks to Mariko Moniwa, Cheryl Peltier,
Virginia Spencer, and Jim Davie (Manitoba Institute of Cell
Biology) for providing histone extracts and H4-acetylated
antibody. We would also like to thank J. F. Mushinski
[National Institutes of Health (NIH), Bethesda, MD] for
providing the Pre-B (abl) cell line. G. M. S. was supported by
the Seller's Foundation (CancerCare, Manitoba, Canada).
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Smith et al.
Neoplasia . Vol. 5, No. 2, 2003


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My friend Mari and I had been discussing how placid our lives have become since school ended, now that we have traded the emotional highs and lows of college life for steady paychecks, steady relationships and predictable routines.
Fortunately, however, you appeared with pleasingly ironic timing to shake us out of our complacency. We were just placidly browsing along St. Mark's Place when you suddenly told Mari and me about that very naughty thing you wanted to do with us. And not just you, but the guy who was working with you, too! Thanks!
We hurried into the store next door, and I thought about you for a few minutes (oh yes, I did). I finally decided to preserve the experience for my Internet scrapbook (i.e. my blog) by taking a photo of you with my handy camera-phone. I don't understand why you looked so surprised. How could I let this precious moment slip by without taking a photo?
I also don't understand why you didn't answer me when I asked you why you said those things to us. After all, you did initiate the conversation, and I genuinely wanted to know. And I don't understand why you got so angry when I mused out loud that it might be because you're an ugly pervert who gratifies himself by harassing random women. After all, I thought you might also want to know how it feels to be treated like a piece of meat.
I think you should be honored to know that you're the first man I've ever snapped back at. Over the three years I've lived in New York City, I've had many strange men do and say extraordinarily nasty things to me. In fact, some of the things they've said or done have been even nastier, and even more degrading. But you, dear sir, are the first.
I've been told that I should just get used to it; that, being a woman and all, I need to take it for granted that I will be harassed on the street. But while I have become a bit complacent, I like to think that I haven't yet mutated into a doormat.
Hugs and kisses (not really),
P.S. By the way, if you're going to sexually harass someone, try not to do it while you're at work. I think that sock store deserves more attentive employees. Plus, with all those frilly anklets in stock, your boss might not be happy if you start scaring away the lady customers.
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Wadena Highway 10 resurfacing project delayed
WADENA -- The microsurfacing project on Highway 10 through the city of Wadena has been delayed due to equipment problems on the job site, MnDOT said Thursday afternoon.
Motorists will travel on the milled surface until it can be paved, early next week. Paving should be completed within two days, weather permitting.
Crews will return to the project in late September to place the permanent striping, once the pavement has had time to cure.
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I would like to be able to fitBounds to a feature when you click on it, but when it zooms in I'd like it to take into account a control layer that will appear once zoomed in, and zoom in but just about 150px to the left. Currently I can accomplish this with the following code, but unfortunately it's not a smooth zoom because my current method will zoom using fitBounds and then once zoomed it uses panBy to pan 150px to the left. This wouldn't be so bad if the panning was smooth, perhaps maybe after a 250ms wait. Ideally I would like to be able to do some math on the bounds passed to the fitBounds method to simply account for the 150px shift to the left.
Here is an example of what I currently have working.
Here is a simplified version of the code I'm using: (you may click here for a fully working version with all of the source code)
when you click
function clickFeature(e) {
var layer = e.target;
map on zoomEnd:
zoomend: function() {
map.panBy([150, 0]);
So, I've achieved the desired function, but it's just not smooth.
Is there a way to just do some math on the bounds that we're getting for the clicked feature so that when we zoom we zoom into an already modified coordinate, thus eliminating the two-step animation process?


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- The Social Platform Solution for the Department of Veterans Affairs
- Technology Insights
- The Cyber Security Report
- The Next Generation Cyber Security Experts
Shows & Panels
- Cloud-Driven Data Insights in Government
- Disaster Resilience for America
- F-35 Lightning II Program
- Government Perspectives on Mobility and the Cloud
- On DoD
- Pentagon Solutions
- Reducing Risk in the Cloud
- Reimagining the Next Generation of Government
- Transforming Business Operations at the Defense Department
Cost doesn't factor heavily in military compensation panel recommendations
The nine-member Military Compensation and Retirement Modernization Commission submitted 15 recommendations to bring effectiveness and efficiency, and that would protect, maintain and improve benefits for the service members.
Panel recommending changes to military health care, benefits
Independent panel recommending changes to military health care, retirement
When is phased retirement coming to your agency?
The waiting game continues for agencies to implement their own guidance and put phased retirement into practice. An OPM spokesman told Federal News Radio it hasn't received any applications for phased retirement so far, and it has no reports on agencies implementing the program.
Phased retirement: Is it coming to your agency?
Julie Perkins, partner at Shaw Bransford and Roth will answer your questions about phased retirement, and Federal Times Senior Writer Andy Medici will give us an update on a new round of legislation that affects federal workers.
January 28, 2015
TSP investors get a bargain in 2014
Retirement plan participants last year paid less than 29 cents in administrative costs for every $1,000 they invested, the Federal Retirement Thrift Investment Board's Kim Weaver told Francis Rose.
Phased Retirement: When? Where? Who?
Agencies are dragging their feet in implementing phased retirement. When they do, how will that affect you? asks Senior Correspondent Mike Causey.
Orphans of the storm
Winter's here and we're all weathering the storm together. What's your blizzard survival secret? Senior Correspondent Mike Causey wants to know.
De-stressing your life
Health and fitness consultant Patti Hudson joins host Bob Leins to discuss what you can do to de-stress your life.
January 26, 2015
Tammy Flanagan, National Institute of Transition Planning
Two congressional updates to the Federal Employee Retirement System are creating different groups of FERS employees. Those are on top of the separation between FERS employees and employees under the Civil Services Retirement System. Tammy Flanagan is Senior Benefits Director for the National Institute of Transition Planning. On In Depth with Francis Rose, she shared her expertise for the first wave of FERS employees to start retiring.
Gridlock and your pension package!
Is congressional gridlock really a bad thing? Maybe not if you're a federal employee or retiree, says Senior Correspondent Mike Causey.
Fearless forecast
NARFE Legislative Director Jessica Klement and Federal Times Senior Writer Andy Medici will predict what's ahead for federal workers and retirees in 2015.
January 21, 2015
New sick leave plan may spotlight fed benefits
Many federal workers probably wonder what's the fuss about giving people seven days of sick leave per year. That's because Uncle Sam has long been ahead of his time when it comes to things like a generous leave package for workers, says Senior Correspondent Mike Causey.
TSP: Is it risky to play it safe?
The G-fund never has a bad day, which is the good news. The bad news, says Senior Correspondent Mike Causey, is that the G-fund also never has a really good day.
Singles and retirement
Gerontology professor and life coach Michael Creedon joins host Bob Leins to discuss the issues and challenges that single retirees face.
January 19, 2015
How are you spending your day?
Just because today is a holiday doesn't mean Uncle Sam has shut down. Far from it, Senior Correspondent Mike Causey says. Lots of people are in today either because they have to be (as in they are essential) or they want to be (as in dedicated). Either way, have a good day ...
3.8 percent pay raise: Reality check!!!
A group of Democrats on Capitol Hill are proposing a 3.8 pay hike for federal employees. That's a good thing, right? Senior Correspondent Mike Causey says the answer is complicated.
Obama authorizes up to six weeks of paid parental leave for feds
President Barack Obama is signing a Presidential Memorandum today directing agencies to provide six weeks of advance sick leave for federal employees to care for their newborn child.
Which TSP fund is the best option for you?
Financial planner Arthur Stein joins host Mike Causey to answer your questions about which TSP fund to invest in.
January 14, 2015
The January effect on your TSP account
You got questions about which TSP fund to invest in? Senior Correspondent Mike Causey may not have all the answers, but he knows someone who does.
House, Senate bills push for 3.8 percent federal pay raise
Companion bills introduced in the House and Senate would give federal employees a 3.8 percent pay raise next year. Federal employees received 1 percent pay raises in both 2014 and 2015, after three years of pay freezes.


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Reflecting on Reflections - June 2014
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Audibility of a CD-Standard A/DA/A Loop Inserted into High-Resolution Audio Playback - September 2007
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AES E-Library
Audio for Multimedia in Russia
A survey on the audio sector of the multimedia market in Russia is discussed. There are many new private companies, mainly spread throughout Russia, which design and produce original audio hardware and software for AT-compatible personal computers. Russian-made audio for multimedia use include both simple PC extensions for education, entertainment, and communication applications; and more complex PC-based digital audio workstations for professional users in the broadcasting, cinema, and recording industries.
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Low Gas Prices are Here to Stay
By Stephen Young
2014 Was a Good Year for LGBTQ Rights
By Stephen Young
The Dumb Bills Proposed to the Texas Legislature
By Stephen Young
Americans Under 30 the Most Cynical Bunch Ever
By Jim Schutze
12 Salads That Don't Suck in Dallas
By foodbitch
13 of the Best Female-Fronted Bands in DFW
By Rachel Watts
Your Baseball Season Guide to Pre- and Post-Game Eats...
By Lauren Drewes Daniels
They're not happy. In fact, there is fear among some in Deep Ellum that the new sewer lines will wind up flushing some businesses down the drain. (Note: That's the last scatological and/or plumbing word play. Promise.)
Or, as Scott Peterson, office manager for Txon Commercial Real Estate puts it, "It's a disaster." Peterson, whose company is a major Deep Ellum landlord, says the city's plans to replace the sewer lines that run behind and under the buildings with new lines under the streets may mean large plumbing bills and worse for some businesses.
The problem is that most businesses placed their sinks and toilets in the rear of their establishments, close to where the old sewer pipes are. Move them, and business owners will be forced to reconnect to the new lines under the streets. Businesses on several blocks along Main, Elm and Commerce--the bulk of Deep Ellum--could be affected, he says.
"It's going to put a lot of people under," Peterson says. Many Deep Ellum bars and restaurants have installed fancy flooring, lights, etc. that will need to be torn up to allow new pipes to be laid. Bars, restaurants and tattoo parlors may have to shut down temporarily.
Putting the new sewer lines where the old ones lie is not financially or technically feasible, and the city can only do work on city property, leaving the tenants and property owners to figure out how to pay for the new plumbing. They'd like the city either to make the connections itself or provide some financial assistance.
Terrace Stewart, director of Dallas Water Utilities, has heard their worries and is working to find some way to make the transition less painful, perhaps by extending the amount of time businesses will have to switch connections to the new lines. State law prohibits spending funds on private property, however, and simply putting the new lines where the old are would be irresponsible, since maintaining them under existing buildings would be tough, if not impossible.
Stewart hopes to come up with some compromise solution before bids for the project are sought, but says the work has to be done, regardless. The sewers under Deep Ellum are some of the city's oldest and, like the beer soaked patrons of Deep Ellum, they've got to go. (Oops. One more.)
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Superman going in the right direction.
Let me start by saying my personal opinion of Superman can be summed up with the word boring. So a 4/5 star from me is actually an amazing credit to the movie. However that could either mean if your a Superman nut you will LOVE it! Or if your a Superman nut you may be able to pick it apart better than I am able to. The only thing that kept me from giving a 5 star rating is I felt the plot was rushed. Even though I do think this is a better movie than the first GI JOE movie I find comparison in that both movies have tons of action that is fun to look at. Both movies however fail to serve up a good plate of meat and potatoes. I wish I could explain myself further, I simply cannot without making this review a spoiler. The movie was definitely worth the price of admission, just not quite worth the cost of the popcorn and soda. It was fun though.


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Puriclean 100g
Puriclean 100gThe Puriclean 100gis superb for use in your caravan or motor home and will clean and purify all stored water systems.
Puriclean 100g : Extra Info
- Handy 100g pack
- 100g will clean up to a 90L tank
- Suitable for tanks, water systems, containers and pipes
How can I get it?
Click & Collect
Delivery available