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New Braunfels Herald Zeitung Newspaper Archives
July 29, 1982
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Thursday, July 29, 1982
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Wednesday, July 28, 1982
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Friday, July 30, 1982
New Braunfels Herald Zeitung
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New Braunfels Herald Zeitung
New Braunfels, Texas
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1952 - 2013
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New Braunfels Herald Zeitung (Newspaper) - July 29, 1982, New Braunfels, Texas Inside Agency considering program expansion By DEBBIE TURNER Staff writer Discussion on proposed expansion, budget approvals and executive session topics dominated the Community Council of South Central Texas (CCSCT) board of directors meeting Wednesday night in Floresville. Six counties — Atascosa. Wilson, Karnes, Frio, Guadalupe, and Comal — were previously funded by the Community Services Administration, Michele Rowcroft, agency executive director, told board members. "At the request of the Texas Department of Community Affairs, expansion is tentatively being considered under block grants for 1983 to the five counties we weathered in 1981," Rowcroft said. Weatherization is a TOCA program, in which home repairs are made in an attempt to stop infiltration of cold and heat, and includes insulation. "This expansion is all very tentative, but TDCA asked us to look at adding Medina. Kendall, Kerr, Gillespie and Bandera counties, along with the six counties already CSA-funded, as CCSCT counties," Rowcroft explained. The expansion move would be a good one in some ways, and costly in other ways, the director added. "However, everything is still up in the air. The TDCA will now send up information letting us know if those five counties are open for us to take on. There's a lot more discussion needed on the subject before we make a decision." A proposed Aging budget of $473,157 for 1983 from the Alamo Area Council of Governments and other private funding sources was approved by the board members, as well as a Family Planning sliding fee scale effective Sept. I, See COUNCIL, Page 14 •frail as, rex a s # vi rile rep Lex, Inc. -ct J Hit ch womb Ie P.O. doz 45 43 6 Comp V-y pq 7^ ^ ll1') Tragic mishap Christian singer among 12 crash victims they apparently did not develop sufficient power to climb or their air speed was too low. It went into the trees ... the trees," said Paul Long, a pilot who witnessed the crash while doing maintenance work LINDALE, Texas (AP) — A twin-engine plane that plunged into a wooded area killing 12 people including eight children and a popular Christian music singer — was designed to carry seven people but that didn't necessarily mean it was overloaded, federal investigators said today. The Cessna 414, leased by the Last Days Ministries here, crashed shortly after takeoff Wednesday night from the ministries' private airstrip near this town about 90 miles east of Dallas. "I can't say it was overloaded until I know the size of the bodies, the amount of fuel, the amount of luggage," said Randy Brooks, a federal Aviation Administration investigator from Shreveport, La. However, Brooks said the manual specified tile plane had seven seats and a toilet seat. Five of the children were four years old or younger. Police and ministry officials said the crash killed Christian music singer Keith Gordon Green, 27, two of his children, the pilot, and eight members of a Simi Valley, Calif., family. The plane was en route to a Dallas airport where members of one of the families were to connect with a commercial flight, said ministry spokesman l^arry DeGraff. FAA spokesman T.C. Miller in Fort Worth said the pilot did not file a flight plan. "The plane rolled down the airstrip and took off For a moment everything appeared normal. But at the airstrip's tower. "All that is left is just a few pieces of burnt metal... just an old burned hull," said Brooks. Warren V. Wandel of the National Transportation Safety Board in Fort Worth said today at the scene that it would be two or three days before his investigation is complete. Cessna representatives were enroute to the site to see if the plane had been modified for more seats, he said. "I don't believe it could ever be modified to seat 12," Wandel said. "The possibility exists, of course. With that many people on board, it could have been overloaded. But we have to calculate a few things before we can say that. Wandel said. The wreckage still smouldered this morning in a gully where it nosedived. Smith County Deputy Larry Willbanks said authorities had a tough time getting through the trees and underbrush to the burning aircraft. "By the time we had. most of the fuel had splattered on the ground so it burned pretty quick," said Willbanks. He said t ie bodies were burned beyond recognition.Reagan to extend Soviet grain sale WASHINGTON (AP) — President Reagan, expected to announce a one-year extension of grain sales to the Soviet Union, says such trade is justified because it drains the Soviets of "hard cash, and they're not too flush with that right now." The president sought to draw a distinction between continued U.S. grain dealings with the Soviets and his opposition to the help Western Europe is giving Moscow for construction of a trans-Siberian natural gas pipeline. Despite strains with the allies over those issues, Reagan maintained that "we do have a fine relationship" with Western Europe. "This is kind of like a fight inside a family, but the family is still a family," he said Wednesday at a nationally televised news conference: Reagan also made a pitch for a constitutional amendment banning deficit spending except with a three-fifths vote of Congress, and said that "slowly and surely we're working our way back to prosperity." He said that acceptance by the Palestine Liberation Organization of Israel's right to exist "would be a step forward," and said he remains hopeful that a political solution will found to end the bloodshed in Lebanon. Reagan's grain-sale decision, which administration officials say will probably be announced before the weekend, was anxiously awaited in the Midwest grain belt. Reagan is expected to extend the existing agreement for one year, even though farmers have pressed for a long-term arrangement. In Denver, Agriculture Secretary John R. Block said a long-term sales agreement with the Soviet Union was "not in the cards at this time." "I would favor that, at least at some point, but not today," Block said. Reagan sought to halt pipeline construction in response to the Soviet role in the military crackdown in Poland. "We have made it clear" that if the military rule is relaxed, "we'd be very happy to review our position with regard to the pipeline," he said. But, the president said, "There are a couple of very important differences" in his support for continued grain sales and his opposition to the pipeline, which would carry fuol from Siberia to Western Europe. "The technology for the pipline is mainly only obtainable from the United States," he said. "Grain, the Soviet Union can get in other places, if they want it." Refen ing to the Carter administration's refusal to ship grain I Hie net Union in the wake of '.he Soviets' militan move into Afghanistan in December 1979, Reagan said We w ouldn't be achieving very much if we had used" such an embargo. "It didn't hurt the Soviet Union, but it was a terrible economic blow to our farmers." He sui I the aik of "grain will result in the Soviet Union having to nay out hard cash, and they're not too flush with that right now." The pipeline, when finished, will result in the Soviet Union getting hard cash, which it does net now have and which it can then use to further build up its military might," the president said. Reagan opened the newrs conference, hi*- 12th since taking office 18 months ago, by saying, "We've begun to rescue this economy and the first evidence of recovery has been sighted, but it's only a beginning." "Many of our people are still suffering and nothing has been more painful to me than the slowness of our progress," he said. The economy is in a period of transition and "we're going to see an improvement in the second half of this year," he said. Once, a reporter asked Reagan, whose 1983 budget incorporates a deficit greater than $100 billion, whether the president's support for the balanced budget amendment was akin to his saying "there ought to be a law against what I am doing." The president responded: "I See REAGAN, Page 14 Goodwin shifts to grade units By DYANNE FRY Staff writer Students returning to Goodwin Primary School this fall won't just be facing a longer day. They'll find their classes a bit reorganized. Three "mixed" units, each composed of some kindergarten, some first-grade and some second-grade students, will be replaced by three single-grade units. "It'll be more like most traditional schools would be," said principal Randy Dry. "The only difference is that we're in a (teacher-)teaming situation, instead of individual classrooms. "We're not 'doing away wrth any of the good things we've been doing," Dry added. "We'll continue to emphasize continuous-progress learning, and will keep the program as individualized as possible." Teachers will be grouped into teams of four and five. As in the past, these teams will divide each unit into basic "skill groups" to work on specific subjects. Under the new plan, "second-grade teachers can spend more time on second-grade skills," said Dr. Victoria Post, assistant superintendent in charge of curriculum for the Comal Independent School District. Math, reading and language arts will be emphasized. Post helped work out Goodwin's new system. But Dry said, "This is an idea that our faculty has kicked around for some time now." His campus began working with multi-grade units in 1974, and he thinks teachers have handled the system well. "But we always had some reservations about putting kindergartners and second-graders together," said Dry. Some teachers thought the older students weren't concentrating enough on academic subjects, and that kindergartners might be getting too heavy a dose. "We're hoping it won't be quite the culture shock when they go to third grade," Dry said. He has the only "primary school" in the district. After second grade, his students graduate to Frazier Elementary, where they attend third, fourth and fifth. CISD's other elementary campuses < Bulverde, Mountain Valley and Comal) each serve grades K through 6, and teaching teams have generally worked with just two levels at a time. Frazier established a separate third-grade unit last term. This year, it will be split into three single-level groups, just as Goodwin will. Bulverde has been moving in the same direction, Post said, and this year their students, too, will be divided differently. "It's a better use of teachers for the larger campuses," she said. If there are enough teachers at one level to work with fast, medium and slow skill groups, Post feels ifs more efficient to keep teachers in the grades they were trained for. In smaller schools like Mountain Valley and Comal, that isn't always possible. Post said. Multi-level units will probably continue there to some extent. A longer school day fits right in with Goodwin's new emphasis on academics. Dr. Edgar Millheim, CISD's new superintendent, set a minimum number of minutes per day to be spent on the three basic subjects. Goodwin's old schedule, with the last bell at 2:50 p.m., didn't allow enough time for all the necessary activities, Dry said. The new release time, 3:35 p.m., will coincide with Frazier's. The new class system, means that some students will have a different advisory teacher this year. Parents should call the school at 625-2822 on Aug. 17, 18 or 19 for teacher assignments, Dry said. Primary students new to ('ISI) should register on the above dates. Teenage traumas Teen agers don't much care for parental advice, but they need it nonetheless, as Steven Levenkron explains in part 3 of the series "How to Survive as a Parent." Page 3. CLASSIFIED.......................11-13 COMICS............................10 CROSSWORD........................10 DEAR ABBY..........................2 DEATHS.............................2 HOROSCOPE.........................2 KALEIDOSCOPE.......................6 OPINIONS............................4 SCRAPBOOK.........................6 SPORTS...........................7, 8 STOCKS............................14 TV LISTINGS.........................IO WEATHER...........................2 THURSDAY July 29,1982 25 cents (USPS 377-880) d-Zeituna 14 PagesMcKenna expansion groundbreaking due in August By JACQUELINE SMITH Staff writer Hospital officials have their eyes on the calendar looking for a special date. That date, which will be determined within the next week, is something they've been looking forward to for a long time — the groundbreaking date for the expansion of McKenna Memorial. Hospital administrator Tom McNeal thinks the groundbreaking will be held within the next couple weeks. "It could be as soon as the second week of August sometime," he said Thursday. One thing which cleared the way for the groundbreaking date to be set was the 20-month construction contract unanimously approved Wednesday by the hospital's board of directors. This contract, with Clearwater Constructors Inc., of Austin, was one of ll construction bids sub-mi tteed. Clearwater's bid of $6,490,000 was ti*' lowest one. At the time the bids came in (in early Juno McNeal and the hospital's architects Jason Frye Architects Incorp. of Houston, were quite surprised at both the number and dollar amount of bids submitted. They were "especially delighted" to discover that Clearwater's bid was $310,000 lower than anticipated. The two second lowest bids, which amounted lo $6,845,000 and $6,919,000, were submitted by El See HOSPITAL. Page 14Thompson getsfive-year sentence New Braunfels, Texas Vol. 91 — No. 148 Floating trash Litter is one of the unpleasant signs of summer in Comal County. The aesthetics of Staff photo by John Senter this Comal River scene is a jarring contrast to th<> floating bottle and V 8 can. A San Antonio man who pleaded guilty to two counts of involuntary manslaughter in connection with a fatal traffic accident iii Comal County last November has been sentenced to five years iii the Texas Department of Corrections on each count. Merrill J. Thompson pleaded guilty July 20. He received the two live-year sentences from 207111 District Court presiding judge Robert Pfeuffer. They will run concurrently, District Attorney Bill Schroeder said. Formal sentencing has been scheduled for Augsut 19. See THOMPSEN, Page 14 New Braunfels ;
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AnalogClock White
by: Black Bear Blanc • 6
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by: Black Bear Blanc • 6
UCCW 2.1.2 or higher is required: http://goo.gl/TZgw0
Uccw skin is skins for widgets made with the app UCCW (Ultimate Custom Clock Widget).
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Coronado 60mm SolarMax II Double Stack Solar Telescope Kit (10mm Blocking Filter)
- 60mm Solar Viewing Refractor
- 400mm Focal Length, f/6.6 Focal Ratio
- Double-Stacked Filter System
- 10mm Blocking Filter
- RichView Tuning Assembly
- 0.5 Angstrom Bandpass
- Sky-Watcher AllView Mount
- Motorized Computer-Guided Go-To Mount
- 42,900 Object Database Hand Controller
- Adjustable-Height Steel Tripod
B&H assembled the 60mm SolarMax II Double Stack Solar Telescope Kit to give users a complete observation platform with which to safely view the Sun. The kit includes the Coronado SolarMax II 60mm Double Stack Solar Solar Telescope and the Sky-Watcher AllView computer-controlled motorized mount.
- Coronado SolarMax II 60 60mm f/6.6 H-alpha Double-Stack Solar Telescope (10mm Blocking, OTA Only)
Coronado 's versatile SolarMax II 60 60mm f/6.6 H-alpha Double-Stack Solar Telescope is calibrated for the hydrogen-alpha wavelength, and comes equipped with their RichView tuning system. This system allows direct adjustment of the primary etalon filter enabling it to be tuned up or down from the 6562.8? wavelength for highlighting specific zones of the chromosphere and to compensate for Doppler shift that can pull prominences and other features off-band and affect views. Tuning areas of concentration back on-band, allows for higher-contrast views of prominences on the solar limb.
Its primary internal etalon provides a bandpass of 0.7? which will reveal prominences in high contrast. Prominences generally appear larger with this size filter than in narrower bandwidths. This version of the SolarMax II comes with a secondary etalon that fits on the front of the OTA and narrows the bandpass to 0.5 Angstroms. At this bandpass, surface contrast is improved versus wider bandpasses. This ability to change the bandpass allows users to see more areas of the Sun than with a single-etalon solar scope. Complementing the etalon filters is a rear 90° 10mm blocking filter. This secondary filter further isolates the H-alpha wavelength for better views. While a smaller blocking filter would suffice, Coronado includes this larger option to allow the use of eyepieces with larger field-stop diameters to be used without vignetting occurring, as might be seen with smaller filters, which would interfere with the field of view
The SolarMax II is offered here without a mount or tripod, but comes with a wide clamshell mounting ring that is outfitted with a 1/4"-20 socket for attachment onto many photo/video/field tripods or on piggyback rigs, or an optional Vixen-style dovetail plate is available for use with conventional alt-az, EQ, or motorized telescope mounts.
- Optics
- 60mm aperture
- f/6.6 focal ratio
- 400mm focal length
- Refractor-style lens configuration
- Solar Filter
- Designed for viewing in the hydrogen-alpha wavelength (6562.8?)
- Versatile etalon configurations enables easy viewing of both surface detail and prominences
- Internal primary etalon blocking filter provides a 0.7? bandpass
- Narrower 0.5? bandpass when using included front secondary etalon
- Rear 10mm diagonal secondary blocking filter allows for the use of eyepieces with larger field stop diameters without vignetting in the field of view
- Proprietary RichView tuning system enablesthe internal etalon to be tuned for Doppler compensation or to highlight specific zones
- Thermal Stability: 0.005?/°C
- Included Cemax Eyepiece
- 1.25" diameter barrel
- 25mm focal length yields 16x
- 52° field of view
- Fold-down rubber eyecup for use with or without eyewear
- Designed specifically for use at H-alpha wavelengths
- 20mm eye relief
- Use and Handling
- 1.25" diameter drawtube
- Helical focuser with easy-to-use focus collar
- Sol Range solar viewfinder enables safe aiming of the OTA at the Sun
- Included carrying case keeps OTA safe during transportation and storage
- Mounting
- Mount and tripod required
- Mounting ring allows the relative position of the OTA to the mount to be adjusted for comfort and limit obstruction
- Clamshell mounting ring with 1/4"-20 mount for using photo/video/field tripods or piggyback rigs
- Optional Vixen-style dovetail plate available for use with conventional telescope mounts
- Sky-Watcher Sky-Watcher AllView Mount
The Sky-Watcher AllView Mount from Celestron is a multi-purpose mount designed to be used as an altazimuth go-to computerized astronomical mount (using an optional telescope optical tube), a motorized platform for taking panoramic photographs, a stable platform for panning while taking time lapse video or still images, and a motorized base for smooth panning video.
It comes with a multi-purpose mounting bracket for attaching an optical tube, still camera, or video camera. Dual optical encoders make sure that the mount never loses position even when moved by hand. It is supported by a sturdy stainless steel tripod with an accessory tray. The hand controller accesses a database of 42,900 astronomical objects. An electronic shutter release cable for most Canon DSLR cameras is included while other optional cables are available for alternate cameras.
Not only can the Sky-Watcher AllView Mount be used as a computer controlled telescope mount, it can also be used as a motorized tripod for photography or video recording as well. The AllView mount and the panoramic functionality of the SynScan AZ hand controller work together as a panoramic controlled mount and head. There are three methods for shooting panoramic images - Easy Pano Mode, Preset Pano Mode and Time Lapse Mode. The AZ GOTO MODE provides extensive computerized go-to functionality to assist in finding and enjoying the treasures of the night sky. The user-friendly menu system allows automatic slewing to over 42,900 objects.
The SynScan AZ hand control has three ports on its bottom edge. The hand control cable has a RJ-45 with 8 connecting pins on one end and a RJ-12 connector with 6 pins on the opposite end. The center port on the bottom of the hand control is used for RS-232 communications between the SynScan AZ and a computer or other device. The DC power port allows independent use of the SynScan AZ hand control for users who wish to browse the database or update the firmware without connecting to the optical device.
- Leveling bubble provided on the mount's base to check the mount's level
- Mounting bracket can be configured to hold a camera in landscape or portrait orientation
- ¼-20 mounting screw on the Landscape Mounting Plate to attach camera, camcorder or telescope optical tube
- Dual-Encoder design on both axes of the mount means that the mount will not lose its alignment or positional information on either axis, even if the scope is moved by hand
- Requires 12 Volts DC, or 8 - 16 Volts with an optional AC adapter or ten AA alkaline batteries in the built-in battery compartment
- Mounting bracket assembly includes Dovetail Bar, Guiding Rail, Landscape Mounting Plate and Portrait Mounting Plate
- Time Lapse Mode permits a series of still images over a long period of time while the mount slowly pans
- Video coursing feature allows video with a constant slewing speed from one point to another
- Hand control has a dual-line, 16-character display that is backlit for comfortable viewing
- Three methods to align the SynScan AZ - Brightest Star alignment, 2-Star alignment and Daytime alignment
- SynScan AZ provides a Pointing Accuracy Enhancement (PAE) function to further improve accuracy
- SynScan AZ database with over 42,900 celestial objects includes complete Messier, NGC, IC SAO catalogues and 25 user defined objects and is flash upgradeable
- The Tour presents highlights of what is in the sky at that moment presenting the brightest and most beautiful objects
- Slew Speeds: 1000x, 800x, 600x, 400x, 200x, 32x, 16x, 8x, 2x, 1x
Tracking Rates: Sidereal, Lunar, Solar
Tracking Modes: Dual Axis Tracking
Dovetail Compatible: accepts "Vixen-style" dovetail
Go-To Pointing Accuracy: up to 5 arc min
- Coronado SolarMax II 60 60mm f/6.6 H-alpha Double-Stack Solar Telescope (10mm Blocking, OTA Only)
Aperture 60 mm Focal Length 400 mm Focal Ratio f/6.6 Bandwidth Hydrogen-alpha, 6562.8 ?
Single-etalon: <0.7 ?
Double-stacked etalons: <0.5 ?
Thermal Stability 0.005 ?/°C Blocking 10 mm Finderscope Sol Ranger Mount Clamshell ring with 1/4"-20 mount Length 16.75" / 42.5 cm Weight 6.0 lb/ 2.7 kg Packaging Info Box Dimensions (LxWxH) 28.0 x 11.2 x 9.75"
- Sky-Watcher Sky-Watcher AllView Mount
Mount Type Altazimuth Load Capacity 9 lb / 4.1 kg Motor DC Servo Motors GO-TO Capability Yes Altitude / Latitude Adjustment Yes Polar Axis Scope Alignment method: brightest star, 2-star and daytime Includes Tripod/Pier Adjustable height, steel tripod legs Power Source 8V to 15V DC 1Amp Weight 21 lb / 9.53 kg Packaging Info Box Dimensions (LxWxH) 44.0 x 14.5 x 11.0"
- SolarMax II 60 60mm f/6.6 H-alpha Double-Stack Solar Telescope (10mm Blocking, OTA Only)
- T-Max 60 - Doppler Tuning Unit
- SolarMax 60 Filter
- Cemax 25mm Eyepiece
- 90° 10mm H-Alpha Blocking Filter (1.25")
- Sol Ranger Solar Viewfinder
- Clamshell Mounting Rings
- Travel Case
- Limited 1-Year Warranty
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Fiber Channel (FC)
Definition - What does Fiber Channel (FC) mean?
A fiber channel (FC) is a computer networking technology that is used to transfer data between one or more computers at very high speeds. It was initially designed for supercomputers but is now commonly implemented in storage networking server environments as a replacement to small computer system interface (SCSI) and other serial storage technologies.
Techopedia explains Fiber Channel (FC)
FC is used in a server environment to transfer bulk data between interconnected storage servers or clusters at very high data transfer rates (DTR). It can transfer data in excess of 1 Gbps and reach speed up to 4 Gbps.
FC-based data transfer is usually achieved by using a FC port on a computer or server and a FC-specific switch, which is known as the fabric. The port and switch can be connected using standard coaxial cables or through fiber optic cables.
The signals transmitted from a FC port can be propagated to substantial distances, reaching several kilometers in length with high-speed mediums.
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My den: it's my sanctuary
It's a quiet barrio just to 30 minutes from down town, Capital Federal, Buenos Aires. Good neighbour. It is close to a big park where a lot of people go running and biking and take sun. You have a little comercial center very close where to buy groceries and vegetables etc.
Galleries/Museums, Cultural/Tourist Attractions, Children's Activities, Public Parks, Restaurants/Bars, Clubs, Serious Shopping, Theatres, Spa, Health Club, Tennis, Golfing, Outdoor Biking, Beach/Swimming, Boating/Sailing, Surfing/Windsurfing/Kitesurfing, Canoe/Kayaking, Horseback Riding
Take the train and know this very big and beautiful city
To organize a home exchange, you need to be a member


text:
From Eden to Exile : The Epic History of the People of the Bible Paperback
by David Rohl
As the leading figure in New Archaeology, David Rohl has been at the forefront of the movement to discover the archaeological evidence for events described in the Old Testament which we have come to think of as myths.
His previous books, Test of Time and Legend presented the arguments and counter-arguments.
In From Eden to Exile this discursive approach is replaced by historical storytelling, which follows the sequence of events from the rise of Neolithic civilisation in a region now part of Iran which inspired the story of the Garden of Eden, through Noah, Abraham and the sojourn in Egypt, to the fall of Jericho, the dual kingdoms of the Promised Land and lastly, the exile in Babylon, where the stories of the Old Testament were collated into something very like their present form.
A fascinating history book that will appeal to anyone with an interest in ancient history, religion and archaeology.
- Format: Paperback
- Pages: 528 pages, Illustrations, maps
- Publisher: Cornerstone
- Publication Date: 02/10/2003
- Category: Biblical archaeology
Free Home Delivery
on all orders
Pick up orders
from local bookshops


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ED Magedson – Founder
Ripoff Report
sears and contractor29807 Wolfe Rd, Circleville, Ohio 43113 Internet United States of America
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The unfortunate reality is that I quickly dislike (and subsequently don't finish) a majority of the games I own. (That most of these games are critically acclaimed and boast mass appeal is merely a twisted curiosity.) I do, however, try to keep an open mind for as long as possible, but most games just get... boring. This said, I very rarely am guilty of not giving games sufficient time to grab and compel me to see them through. Indeed, it took 21 acts of Uncharted 2 (hated it after the fourth or fifth, though), five hours of the first Uncharted, and 30 minutes of inFamous--just to name a few examples--to utterly annihilate my will to complete them.
(Now, you're probably questioning my short leash for inFamous, but you can't blame me for not wanting to spend even MORE time on Spiderman 2, can you?)
Anyway, I tried to create a formula that would accurately explain what keeps me playing a video game. But what I conjured didn't always fit. Indeed, I simplified it to the convergence of a few simple cogs of interest; a perfect storm of design triumphs, if you will. But the formula didn't explain my completion (and thorough enjoyment) of games that hooked me with a singular aspect. Take Max Payne as a for instance. It wasn't a conglomeration of its story, characters, and atmosphere that kept me coming back; it was the bullet-time. And it wasn't Final Fantasy XIII's visuals and narrative, but the simple joy of developing my characters via the ground-breaking battle system that trapped me.
This brings me to Gears of War. I've had a 360 since launch, but never attempted a go at the series because it looked incredibly, for lack of a more appropriate word, dumb. I recently started playing it, however, and am embarrassed by the fact that I'm now discovering how great it is. And it isn't the story, Marcus and the rest of Team Delta's meat-headed machismo (although they are quite entertaining and not nearly as annoying as they look--especially Marcus), or the visuals (which still hold up quite well despite being nearly five years old) that won me. It's the frantic firefights that are made all the more sweeter by the Hardcore difficulty setting that's not letting me go. I have the Triple Pack so I'm going to be thoroughly caught up by the time 3 is released.
Why does Ninty release things on Sunday? Even with next day shipping, my OoT3D hasn't arrived yet because, according to Amazon, "UPS does not work on Sundays!!"
And I curse you, UPS, for always delivering my packages at 7:00 PM and not a minute sooner.


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Morphological and functional data indicate that glutamatergic innervation of the hypothalamic paraventricular nucleus plays an important role in the control of this prominent cell group. Sources of this neural input are unknown. The present investigations were aimed at studying this question. The retrograde tracer [3H]D-aspartate, which is selectively taken… (More)
- Hajnalka Bokor, Agnes Csáki, Katalin Kocsis, József Kiss
- The European journal of neuroscience
- 2002
Little is known about the neurochemical features of the nucleus reuniens thalami (RE). In the present study, immunocytochemical experiments were performed to characterize the expression pattern of certain neurochemical markers, e.g. the calcium-binding proteins calbindin and calretinin and several neuropeptides. Colocalization studies revealed that half of… (More)
- József Kiss, Agnes Csáki, Zsolt Csaba, Béla Halász
- The European journal of neuroscience
- 2008
The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore,… (More)
- J Kiss, A Csáki, H Bokor, M Shanabrough, C Leranth
- Neuroscience
- 2000
It is well established that the supramammillary nucleus plays a critical role in hippocampal theta rhythm generation/regulation by its direct and indirect (via the septal complex) connections to the hippocampus. Previous morphological and electrophysiological studies indicate that both the supramammillo-hippocampal and supramammillo-septal efferents contain… (More)
The medial preoptic area is a key structure in the neural control of reproduction. Considerable evidence has accumulated indicating that glutamatergic innervation of the area plays an important role in this control. Sources of the glutamatergic input are unknown. Present investigations were aimed at studying this question. [3H]D-aspartate, which is… (More)
The supramammillary neurons projecting directly to the hippocampus or indirectly via the septum participate in the regulation of hippocampal theta activity. Inputs to the supramammillary nucleus are only partly specified neurochemically. Glutamate appears to be an excitatory transmitter in this cell group, however, the origin of the glutamatergic afferents… (More)
The medial preoptic area is a key structure in the control of reproduction. Several data suggest that excitatory amino acids are involved in the regulation of this function and the major site of this action is the medial preoptic region. Data concerning the neuromorphology of the glutamatergic innervation of the medial preoptic area are fragmentary. The… (More)
- V Vereczki, K Köves, A Csáki, K Grósz, G E Hoffman, G Fiskum
- Neuroscience
- 2006
In our present work utilizing the retrograde or anterograde transport of tracers (biotinylated dextran amine and Fluorogold, respectively) we have provided direct evidence for the cells of origin of the limboretinal pathway in rats and their termination in the retina using light microscopic approach. Administration of biotinylated dextran amine into the… (More)
Oxytocin and vasopressin neurosecretory neurons of the supraoptic nucleus receive a rich glutamatergic innervation. The nerve cells of this prominent structure express various ionotropic and metabotropic glutamate receptor subtypes and there is converging evidence that glutamate acts as an excitatory transmitter in the control of release of oxytocin and… (More)
- József Kiss, Zsolt Csaba, Agnes Csáki, Béla Halász
- The European journal of neuroscience
- 2005
Abstract The hypothalamic arcuate nucleus contains a number of neurochemically different cell populations, among others neuropeptide Y (NPY)- and pro-opiomelanocortin (POMC)-derived peptide-expressing neurons; both are involved in the regulation of feeding and energy homeostasis, NPY neurons also in the release of hypophysiotropic hormones, sexual behaviour… (More)


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STEVENS FOULS OUT IN VEGAS LAS VEGAS (Oct. 23, 2005) - Richie Stevens Jr. was well-rested, relaxed and ready to go this morning in the first round of Pro Stock eliminations at the ACDelco Las Vegas NHRA Nationals at The Strip at Las Vegas Motor...
LAS VEGAS (Oct. 23, 2005) - Richie Stevens Jr. was well-rested, relaxed and ready to go this morning in the first round of Pro Stock eliminations at the ACDelco Las Vegas NHRA Nationals at The Strip at Las Vegas Motor Speedway.
Unfortunately, the gremlins he's been battling all year took over again as he red-lighted for the third time this season, losing the opening round to Greg Stanfield, who took the victory with a 6.866-second ET at 201.31 mph.
Stevens' potentially winning 6.860/201.16 pass was disqualified.
"I didn't even know I red-lighted until I got to the end," said the clearly disappointed driver of the Team Mopar/Oakley Dodge Stratus R/T.
"I waited a second (at the starting line). I wasn't going to go right in because I knew he likes to stage last. So, I waited a second, I went in and I was ready. I didn't anticipate it. I thought I just got a good light. I left and I was ahead of him a little bit and I thought, We got him.
"We went through to the end and my win light didn't come on. I had no idea I had red-lighted. No idea.
"I just can't believe the way it's gone. We made four good runs in qualifying and I was hitting the tree good.
"I was fine. I went up there with a clear head. I went to bed early last night, had a good night's sleep, woke up fine, everything was good, no problems.
"I might have been up there staged a little deep," he reflected. "I was that much closer to breaking the beam. I will look at the video later. That's the only thing I could think of, because I saw yellow and I went. Just the normal routine.
"But the way my luck has been going lately, it went red, not green."
Stevens fell to ninth in NHRA POWERade Drag Racing Series Pro Stock points, with 1008. His teammate Jeg Coughlin, who went out in the second round, is eighth, with 1029.
Next up is the Automobile Club of Southern California NHRA Finals in Pomona, Calif., Nov. 3-6, which will close out the 2005 NHRA POWERade Drag Racing Series.


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A two bedroom, 2 bathroom Apartment for sale in El Verger located only 400 metres from the beach. The apartment is accessed via a secure gate, leading to private garage and lift to first floor. The apartment itself consists of a bright, open hallway leading to a lounge/dining room, balcony overlooking the pool and gardens, two bathrooms, two bedrooms, fully fitted kitchen and utility area. Private parking and store. There is hot and cold air conditioning throughout the apartment which is to be sold fully furnished. An immaculate property in a great area with viewing highly recommended.
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|Publication number
|Publication type
|Publication date
|May 13, 1969
|Filing date
|Jun 23, 1966
|Priority date
|Sep 29, 1965
|Also published as
|Publication number
|Ichiro Haruna, Norman R Pace, Solomon Spiegelman
|Original Assignee
|Univ Illinois
|Export Citation
|BiBTeX, EndNote, RefMan
|Non-Patent Citations (1), Referenced by (12), Classifications (17)
|External Links: USPTO, USPTO Assignment, Espacenet
May 13, 1969 s. SPIEGELMAN ETAI- 3,444,042
PURIFIED REPLICASES AND THEIR USES Sheet Filed June 23, 1966 awhqmommoo Emu \REPLICASE so e tmzmo 0mm 0mm FRACTION NUMBER IIVVENTORS SOLOMON SP/EGELMAN lCH/RO HARU/VA v NORMAN R. PACE B) QbJ A/L L q r-1Q ATTORNEYS May 13, 1969 smEGELMAN ET AL 3,444,042
PURIFIED REPLICASES AND THEIR USES Filed June 23, 1966 Sheet of 6 3000- I AFTER c cl $9 Q LU Di 0 2000 0.. (I 0 2 BEFORE c cu E lOOO Q. U
v i 2 i Y RNA ADDED AFTER c Cl 5 7 Q [U (I 2 BEFORE c c| C! 0 U *E g IO3 U T I I I I 4O 6O I00 120 I40 TIME (minutes) NVENTORS SOLOMON SP/EGELMAN lCH/RO HARU/VA NORMAN R. PACE M,ZLWZM;JZ7M leg ATTORNEYS May 13, 1969 s, sPlEGELMAN ET AL 3,444,042
PURIFIED 'REPLICASES AND THEIR USES Sheet Filed June 23, 1966 FRACTION NUMBER S M m M W T L s, Nana P P W m R NH v 0 Mami 0 0R L R 1 E 3 0 H 0 M SMN w m l m 5 m. 2 R M m l 1 z n w W M N .0 A\\\!..o| 5 U M I M lollrlllllllli? F E o s l r m. e m C 5 5553 333.3 m n-v m w w owQmoamooz E8 ATTORNEYS May 13, 1969 Filed June 23, 1966 s. SPIEGELMAN ET AL 3,444,042
PURIFIED REPLICASES AND THEIR USES Sheet 5 of6 IOOO 9 PRODUCT H3 op MARKER FRACTIONS IN VEN TORS $0LOM0/V SP/EGE LMAN lCH/RU HARU/VA NORMA/V R. PACE %W, 2 Ma/Z-Q.e a27f -0 191 ATTORNEYS May 13, 1969 Sheet Filed June 23, 1966 856M526 zm an o 0 w w m m w m 3 2 m a i m M 0m E O H R W D C P R E E P M 5 F S l H 2 N l N R o IN 0 0 D x U N W\ Y. M M x A N M B W M S .0 P W W A N 0 U m m w l 5 T U W m 0 n 0 5 10. x 3 mN O\w. zD mDOCOmmZ: N Nu m w m w 7 6 5 AM 3 2 l 6 7 B 9 IO U TRANSFERS ATTORNEYS 3,444,042 PURIFIED REPLICASES AND THEIR USES Solomon Spiegelman and Ichiro Haruna, Champaign, and
Norman R. Pace, Urbana, Ill., assignors to University of Illinois Foundation, Urbana, III., a corporation of Illinois Filed June 23, 1966, Ser. No. 559,933 Int. Cl. C08b 19/00; C12k 1/10; ClZd 13/10 US. Cl. 19528 17 Claims ABSTRACT OF THE DISCLOSURE Biologically active nucleic acids are synthesized in vitro from nucleotide bases and a purified replicase free of detectable destructive contaminants and substantially free of viral infectivity.
A United States Government contract or grant from or by the Public Health Service supported at least some of the work set forth herein.
This invention relates to methods and systems useful in the synthesis in vitro of biologically active nucleic acids and the isolation of such biologically active nucleic acids, the preparation of purified enzymic components of these systems, the resulting purified enzymic components which are effectively free of detectable levels of degrading enzymes and enzyme inhibitors, and substantially free of infective viral particles and enzyme inhibitors, and the biologically active nucleic acids produced therewith.
As used herein, the term biologically active includes the basis for the assay procedure described, infra, namely, the production of infectious viral RNA, and, more generally, includes materials that possess genetically competent characteristics or information essential to life or processes thereof. These biologically active materials are genetically competent and can transmit information to asystem that will follow their instructions and translate them into biological sense.
Living organisms, including humans, animals, plants, and microorganisms, use biologically active nucleic acids in the processes of storing and transmitting translatable genetic or hereditary information or messages and in the synthesis of the large number of tissue and body proteins. Two nucleic acids which can function under proper conditions as transmitters of the genetic code are DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). In the living organism, these nucleic acids are generally combined with proteins to form nucleo-proteins.
These DNA and RNA molecules consist of comparatively simple constituent nucleotides (nitrogen base, pentose sugar moiety, and phosphate groups) polymerized into chains containing hundreds to thousands of these nucleotide units generally linked together through chemical bonds formed between the constituent phosphate and sugar groups.
These nitrogen bases are classified as purines or pyrimidines. The pentose sugar is either ribose or deoxyribose. Phosphoric acid groups are common to both DNA and RNA. On complete hydrolysis, DNA and RNA yield the following compounds:
Phosphoric acid Phosphoric acid ice It should be noted that the bases adenine (A), cytosine (C), and guanine (G) are comon to both DNA and RNA; the base thymine (T) of DNA is completely replaced by the base uracil (U) in RNA. Methylcytosine occurs in small amounts in various deoxyribonucleic acids of animal origin and in wheat germ. In the DNA of several bacteriophages, cytosine is completely replaced by hydroxymethylcytosine.
Hydrolysis of these nucleic acids under appropriate conditions liberates a group of compounds known as nucleotides; these nucleotides consist of a purine or pyrimidine bases linked to pentose sugar moiety, which sugar moiety is esterified with phosphoric acid. These nucleotides are the subunits from which polymeric nucleic acids are constructed.
The ribonucleic acid polynucleotide structure may be represented diagrammatically, for example, as follows:
pentose phosphate pentose phosphate pentos e phosphate The dotted lines above represent ester groupings between one of the free hydroxyl groups of the pentose and of the phosphate groups. The subscript n represents the number of repeating units which constitute the particular ribonucleic acid molecule.
Recent studies by chemists have shown that the DNA molecule has a doubly stranded chain which, when shown in three dimensions, has two chains intertwined in a double helix. Each chain consists of alternating nucleotides, there being ten nucleotides in each chain per rotation of the helix, this ten nucleotide chain being about 3-4 A. in length. Both chains are right handed helices. These helices are evidently held together by hydrogen bonds formed between the hydrogen, nitrogen, and oxygen atoms in the respective chains. The structure of the DNA molecule as it relates to the sequence of these bases in the molecule is now being elucidated; these structural studies are important, since it is now generally believed that this sequence of bases is the code by means of which the DNA molecule conveys or transmits its genetic information.
Chemists have shown that RNA generally is a singlestranded structure that has in its backbone the S-carbon sugar ribose instead of the S-carbon deoxyribose sugar found in DNA. As in DNA, the different nucleotides are linked together through the phosphate groups to form a long chain and thus to form an RNA molecule of high molecular weight. The RNA molecules do not seem to be as highly polymerized as the DNA molecules, and although there is evidence of hydrogen bonding between the RNA bases in some viruses (e.g., reovirus), it is thought that no helical structure is involved. As with DNA, base sequence studies are now being made with RNA.
In genes, the repository of hereditary factors of living cells and viruses, specific genetic information resides in the nucleotide sequence appearing in the DNA and RNA molecules. These sequences are transmitted, encoded and reproduced in vivo by the complex enzymic systems present in living organisms. If no modification of the genetic DNA or RNA takes place, an exact duplicate or replicate of the nucleotide sequence is produced; this newly formed RNA or DNA in turn results in the production in vivo of an exact duplicate or replicate of a particular protein molecule. If, however, a change takes place in the DNA or RNA molecules, which change can be mediated by some mechanism such as radiation, a foreign chemical reactant, etc., a mutation takes place wherein the altered DNA or RNA molecules duplicate or replicate the new DNA or RNA and these in turn produce new or altered proteins as dictated by the altered nucleotide structure.
Copending application Ser. No. 535,596, filed Mar. 18, 1966, which is a continuation of application Ser. No. 509,458, filed Sept. 29, 1965, now abandoned, discloses a method and controlled system for synthesizing in vitro biologically active nucleic acids using an initiating amount of intact, biologically active (genetically competent) nucleic acid template, replicase and the requisite nucleotides. With this method, one may synthesize, for example, a ribonucleic acid molecule (RNA) identical with the intact template continuously over extended periods until or unless one arbitrarily or selectively stops the synthesis. This self-replication involves the true and complete transmission and translation from the intact template to the nucleotides, whereby the nucleotides are assembled structurally in the identical sequence that characterizes the intact template. The intact product synthesizcd may be either selectively labeled (e.g., radioactive) or nonlabeled and be in a form that is free of detectable impurities or other materials with which it is otherwise found in nature.
More specifically, application Ser. No. 535,596, now pending, discloses a controlled system that provides for the synthesis of intact, biologically active nucleic acid in a buffered aqueous in vitro enzymatic system from nucleotide bases using a selected, intact, biologically active nucleic acid free of detectable levels of destructive material as a template (e.g., input template). When the system produces biologically active replicas (identical copies of the same molecular weight) of the nucleic acid. template, the process is referred to as one involving replication. The enzyme catalyst may be referred to as a polymerase" or replicase; when the enzyme catalyst is an RNA-dependent RNA-polymerase, it is defined as a replicase.
The process or system of the pending; application is particularly well suited for synthesizing in vitro biologically active ribonucleic acid (RNA) from ribonucleotide base components (substrates) having high bond energy, using an intact, homologous (contains the information for its specific replicase) biologically active RNA template, a homologous replicase that selectively recognizes the structural program or message of the template, has catalytic activity for the synthesis of intact biologically active RNA from ribonucleotides, and is effectively free of detectable levels of ribonuclease activity and detectable levels of other destructive enzymological activity, and using divalent ions (Mg++) as a cofactor. The replication process may be stopped by a number of procedures, the simplest of which involves the cooling of the reaction to a temperature at which the rate of enzymic activity becomes negligible, e.g., C.
The replicase for viral RNA can be obtained either by introducing a selected virus nucleic acid (e.g., bacteriophage) free of any existing protective proteinaceous coat into an uninfected host bacterium cell to synthesize an enzyme which is thought not to preexist in the host cell, or, preferably, by introducing an intact bacteriophage (virus particle) into the bacterium cell to synthesize this enzyme.
The injected or intruding viral RNA has a structural program that defines a message that is translated into enzyme protein and is conserved during this translation. This enzyme, a homologous replicase (RNA-dependent RNA-polymerase), is isolated from the altered cell and is then purified to remove detectable levels of the usual concurrent ribonuclease activity and other destructive 4 and confounding enzymological activity which is found in the bacterial cell.
The resulting partially purified enzyme, replicase, discriminately recognizes the intact homologous RNA genome of its original and requires it as a template for normal synthetic replication. Thus, the replicase exhibits a unique and selective dependence on and preference for its homologous viral RNA in exhibiting viral RNA- polymerizing (synthesizing and/or replicating) activity. The replicase exhibits the unique and valuable ability to provide the replication of only intact viral RNA and does not provide for the replication of fragments or foreign sequences or incomplete copies of its own genome. The term genome refers to the entire complement of genes in a cell. The genes provide a repository of genetic information for living cells and viruses.
The nucleotide bases or substrate components for viral RNA replication should have sufliciently high bond energy for replication. Satisfactory replication of viral RNA has been achieved with four riboside triphosphates, namely, adenosine triphosphate (ATP), g-uanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP).
In replicating infectious viral RNA in vitro, the pending application discloses purifying two different RNA replicases induced in a mutant Hfr strain of Escherichia coli (Q-13) by two serologically distinct RNA bacteriophages. The enzyme protein preparations were effectively free of detectable levels of interfering ribonuclease, phosphorylase, and DNA-dependent RNA-polymerase (transcriptase). These isolated enzymes (replicases) showed both a mandatory requirement for template RNA and an ability to mediate prolonged and extensive net synthesis of biologically active polyribonucleotide (RNA). The two replicases exhibited a unique discriminating selectivity in their response to added RNA. Under otherwise optimal conditions, both replicases were virtually inactive with heterologous RNA templates, including ribosomal and s-RNA of the host.
The replicase preparations described in further detail below and constituting a part of the instant invention, are substantially free of detectable levels of virus particles and infectious RNA. In addition, the replicase may be purified so as to be substantially free of contaminants such as carbohydrates, lipids, polynucleotides and other proteins.
The purified biologically active RNA polymerase (replicase), which is substantially free of detectable levels of viral infectivity, and the infective RNA produced with our system and method are intact and are free of impurities or materials with which they are otherwise found in Nature. The synthesized viral RNA, for example, is free of the normally occurring protein coating present in the intact viral particle. The controlled RNA product produced with our system and method thus offers the advantage of being useful in experimental, laboratory, and commercial activities where one wishes to use a biologically active RNA that is effectively free of detectable confounding or extraneous materials. Our controlled system also is free of detectable confounding or extraneous materials and thus provides an important means for studying the mechanism by which genetic changes and replication occur in lifes processes and a means of understanding, modifying or changing such processes or mechanisms.
On a practical basis, the availability of our relatively pure replicase will allow the investigator to move into research areas not previously accessible. Thus, we can now proceed to determine such things as the effect of small or large changes in the replicase molecule upon its ability to synthesize RNA, and to determine the change in the biological activity of the RNA so produced by the altered replicase.
Being a protein and, therefore, made up of a series of amino acids, the structure of the replicase can now be studied, and the relation of its structure to the structure of the RNA produced can give important information, vis-a-vis, structure-activity relationships. Since the replicase is a large molecule and subject to varying degrees of hydrolysis by chemical or enzymatic means, it will be of interest to determine the effect of such hydrolysis, whether they be comparatively minor or major, upon the biological activity of the molecule remaining. In addition, the protein molecule can be subjected to varying degrees of chemical change such as acetylation of its reactive amino or hydroxyl groups, halogenation, nitration, or sulfonation; reaction with nitrous acid should convert the free amino groups of the protein to hydroxyl groups, again with some change in activity.
An RNA template of an in vitro replicating system may be formed in situ. If one were, for example, to introduce foreign bases or nucleotides (e.g., analogues of known bases or nucleotides) into our replicating system, a mutant may be formed which would be the biologically active template for replication with those same bases or nucleotides; in such instances, one would be synthesizing mutants in vitro in a known way.
Our discovery of a method to produce a purified biologically active RNA-dependent RNA-polymerase substantially free of detectable levels of viral infectivity and other biologically inactive contaminants should be useful in the study and/or preparation of products with antiviral activity, anticancer activity, and hormone and/r enzyme activity. Research directed toward the preparation of such products could lead to important therapeutic advancements.
It is conceivable to project that an altered replicase might under certain conditions produce an altered RNA which in our system might possibly have altered virus properties or, under ideal circumstances, might have antivirus properties. It may be possible to use this system by perhaps adding a new component to the bacteria-pure, RNA-virus system, which will result in a new replicase, which replicase system can be directed to produce antiviral molecules.
Using the fractionation system discussed below, a purified replicase preparation is isolated. It is known that disease causing viruses commonly include RNA molecules; for example, the viruses Which cause tobacco and tomato mosaic disease, poliomyelitis, influenza, Newcastle disease in poultry, and mumps, among others, are ribonucleic acid (RNA) containing proteins. Our discovery points to the possibility that replicases for each of these RNA viruses could be derived from an appropriate system. The isolation in vitro of such replicases in purified form provides means for the study of the biochemistry of the diseases and in the preparation of vaccines.
With a pure replicase in hand, it is possible to determine its particular amino acid structure. In addition, with the pure RNA in hand, it should be possible to determine the nucleotide sequence in the RNA, as well as its other structural characteristics. Determination of amino acid structure and coding to give the particular RNA nucleotide sequence should be of importance in elucidating amino acid and nucleotide sequence correlation.
The intact viral RNA used by us as initiating template is isolated from purified virus. It is obtained by deproteinizing the RNA with phenol and purifying the RNA on sucrose gradients. It is not obtained from the virus-infected bacteria but from the complete virus particle.
The replicases were obtained by introducing viral RNA into an isolated mutant Hfr strain of E. coli (Q-13).
Using the in vitro system as herein referred to, the template was produced, for example, by a factor of 10 That is, for each molecule of intact template there are synthesized 10 replicas. Further, micrograms (e.g., 3x10 strands) of synthesized viral RNA are made every 20 minutes per 0.25 ml. of reaction mixture.
The in vitro serial transfer experiments in the pending application, which established that the newly synthesized RNA is a self-propagating and biologically competent entity, requires infectivity assays of the reaction mixtures. A technical complication is introduced by the presence of viable virus particles in the replicase preparations. Their chemical contribution to the RNA content is trivial compared to the amounts synthesized. However, because they have a far greater infective efiiciency than free RNA, even moderate contamination with intact particle cannot be tolerated in material either-being assayed for infectious RNA or being used as material for injection into a host. To obviate these difficulties, all synthesized products are purified with phenol and checked for whole virus particles prior to measurement of infectivity. This, however, makes the assay both laborious and cumbersome, precluding its focile use in laboratory studies or as a therapeutic agent.
Separation of virus particles from the viral replicase can be achieved by taking advantage of their disparities in size and density. The QB virus [J. Bacteriol., 91, 442 (1966)] has a molecular weight of 4.2)(10 and a density of 1.43 gm./crn. It was unlikely that the replicase would be as large or as dense. Successful purification of the replicase by size and density generates more than the convenience of eliminating virus particles. The same procedure also removes free RNA, replicase complexed to postulated replicative forms [cf. -Fed. Proceed, 23, 1285 (1964)].
There is described below the further purification of QB-replicase by banding in CsCl gradients followed by zonal centrifugation in linear gradients of sucrose. The resulting enzyme is substantially free of virus particles and behaves as a single component in the fractionation procedures. Its molecular weight (110,000) and density (1.26) precludes association with so-called replicative forms or negative strands. Its ability to respond to QB-RNA by synthesizing infectious copies remains unaltered. The data discourage invoking a cryptic functioning of pre-existent RNA (doubleor single-stranded) in the reaction being studied.
At this point of the purification process, while the enzyme (replicase) is substantially free of contaminating phage particles and other enzymes, contamination by other biologically inactive materials still exists. The percentage of enzyme (based upon measurements of activity) in the product at this point is in the range of about 0.05% to 0.5% by weight. Further purification of the enzyme by removal of nonenzymatic biologically inactive materials is achieved by using one or more of the following procedures: (1) absorption on C7 alumina (aluminum hydroxide gel); (2) isoelectric precipitation; (3) ammonium sulfate fractionation; and (4) adsorption and elution from DEAE cellulose. Such purified preparation retain in their entirety the characteristics of the replicating enzyme, which basic characteristics are summarized below.
The replicase preparations of this invention include the following characteristics:
(1) The replicase preparation is free of detectable levels of contaminating enzymes such as RNAase, phosphorylase, and DNA-dependent RNA-polymerase;
(2) The replicase preparation is free of significant levels of viral infectivity; it is, therefore, essentially noninfectious and carries no significant concentration of contaminating RNA molecules with it;
(3) When sufiicient purification has been conducted to achieve a substantially purified replicase preparation as described above, the replicase has had removed from it substantial amounts of biologically inactive contaminants;
(4) The replicase requires its homologous RNA as a template;
(5) In the presence of this template, the requisite nucleotides, Mg+ and aqueous buffer, the replicase mediates prolonged and extensive synthesis of the RNA.
The replicase preparations of this invention, unlike those described in said copending application, have the characteristics of (2) and (3), above.
When the replicase is isolated from a bacteriophage infected cell the reaction is characterized further by these properties: (1) the enzyme generates a polynucleotide (RNA) of the same molecular weight as the viral RNA; (2) when reactions are initiated with RNA template at concentrations below saturation of the replicase, autocatalytic synthesis of the RNA is observed in the system; (3) the RNA isolated from this reaction system and further purified can, in turn, serve with full effectiveness as a template; (4) the RNA produced by the enzyme in this system is biologically active, being as fully competent as the original or template RNA to program the synthesis of virus particles in protoplasts.
In the following example, there is described in detail a procedure for the purification of the RNA-dependent RNA-polymerase (replicase) obtained from a bacteriophage infected cell. The example is illustrative of our invention. It will be understood, however, that the invention is not necessarily limited to the particular examples, tests, materials, properties, conditions and procedures described therein.
EXAMPLE (A) Methods and materials Catalase (2X crystallized) was purchased from Sigma Chemical Co., and assayed as described in J. Biol. Chem., 236, 1372 (1964). CsCl was the 99.7% pure material from Penn Rare Metals, Inc., Revere, Pa. An unsatisfactorily high CD. was easily corrected by filtration through a cellulose nitrate membrane filter (Schleicher and Schuell and Co., B-6, 27 mm.).
The host and assay organism is a mutant Hfr strain of E. coli (Q-13) isolated in the laboratory of W. Gilbert of the Department of Biology, Harvard University, Cambridge, Mass., by Diana Vargo, formerly an assistant of Dr. Gilbert, now a graduate student of the Department of Microbiology, University of Illinois, Urbana, 111. It has the convenient property of lacking ribonuclease I and RNA phosphorylase [Fed. Proc., 24, 293 (1965)]. Preparation of virus stocks and purified RNA followed the methods of Doi and Spiegelman in Proc. Natl. Acad. Sci., U.S., 49, 353-360 (1963).
(B) Assay of enzyme activity by incorporation of radioactive nucleotides The standard reaction volume is 0.25 ml. and unless specified differently contains the following in moles: tris HCl pH 7.4, 21; magnesium chloride, 3.2 (when included, manganese chloride, 0.2); CTP, ATP, UTP, and GTP, 0.2 each. The enzyme is usually assayed at a level of 40 g. of protein in the presence of 1 g. of RNA template. The standard reaction is run for 20 minutes at 35 C. and terminated in an ice bath by the addition of 0.15 ml. of neutralized saturated pyrophosphate, 0.15 ml. of neutralized saturated orthophosphate, and 0.1 ml. of 80% trichloracetic acid. The precipitate is transferred to a membrane filter and washed seven times with 5 ml. of cold TCA. The membrane is then dried and counted in a liquid scintillation counter as described in Proc. Natl. Acad. Sci., U.S., 50, 905-911 (1963). This washing procedure brings zero time counts to less than 80 c.p.1rr. with input counts of 1X10 c.p.rn. The specific activities of the labeled triphosphates added are adjusted so that with the efficiency employed, 1x10 c.p.m. corresponds to 0.2 moles of the corresponding triphosphate.
(C) Preparation of infected cells The basic medium employed for growing infected cells and producing virus contained the following in grams per liter: NI-I Cl, 1; MgSO.,-7H O, 0.06; gelatin 1 1O- casamino acids (vitamin free), 15; glycerol, 30; to this is added, after separate autoclaving, 7 ml. of 0.1 M CaCl and 10 ml. containing 4 gm. of Na HPO -7H O and 0.9 gm. KH PO Lysates in liter quantities are first prepared to be used for infection of larger volumes of cell suspensions. These are obtained by infecting log phase cultures (OD of 0.25) with a purified phage preparation at a multiplicity of about 5. They are incubated while shaking at 37 C. until lysis is complete and then monitored for titer and purity of the phage. Such lysates can be stored frozen at 17 C. indefinitely and thawed just prior to use. In general, 35 liter quantities of cells are grown up in carboys to an OD of between 0.275 and 0.290. The temperature in the carboys is 34 C. while the temperature of the water bath in which they are immersed is maintained at 37 C. When the cells reach an CD of 0.275, they are infected with virus at a multiplicity of between 10 and 50 and allowed to aerate for mixing for one minute. The areation is interrupted for 10 minutes for absorption, reinstituted, and the incubation continued. At 25 minutes sufficient sucrose and magnesium are added to give final con centrations of 18% and 0.01 M respectively. After another 5 minutes the process is terminated by the addition of crushed ice. The infected cells are harvested in a Sharples Centrifuge and stored at l4 C., at which temperature ability to yield active enzyme is retained for periods exceeding 6 months. Uninfected cells are prepared and stored in the same manner. To provide uniform preparations for enzyme isolation, the infected cells are thawed sometime prior to use and resuspended (20 grams of packed cells in ml.) in a solution containing 0.01 M tirs buffer pH 7.4, 0.001 M MgCl and 0.0005 M mercaptoethanol and 5 ig/ml. of DNAase (deoxyribonuclease). After thorough resuspension with a magnetic stirrer at 4 C., the suspension is divided into convenient aliquots in plastic tubes, frozen, and stored at 14 C.
(D) Preparation of enzyme The following procedure is described for 20 grams of packed infected cells. The frozen cell suspension ml.) is thawed and to this is added 0.5 mg./ml. of lysozyme following which the mixture is frozen and thawed twice, using methanol and Dry Ice as the freezing mixture. To the lysate are added 0.9 ml. of 1 M MgCl and 2.5 g/ml. DNAase (deoxyribonuclease) and the resulting mixture is incubated for 10 minutes in an ice bath. The extract is then centrifuged for 20 minutes at 30,000Xg. and the supernate removed. The pellet is transferred to a prechilled mortar, ground for 5 minutes, and then resuspended in 30 ml. of the same buffer as used for the cell suspension except that the magnesium concentration is raised to 0.01 M to increase the effectiveness of the DNAase digestion. The extract is then centrifuged at 30,000X g. for 20 minutes and the two supernates combined, adjusted to 0.01 M EDTA, ethylene diamine tetraacetic acid (previously brought to pH 7.4), and incubated at 0 C. for 5 minutes. Insoluble proteins appear and are removed by centrifugation at 30,000 g. for 20 minutes. At this stage, a typical active infected extract has an OD of between and 180. Lower values commonly signal a poor infection with a resulting low yield of enzyme. To the cleared supernatant fluid is added 0.01 mg. of protamine sulfate for each OD unit. After 10 minutes the precipitate, containing virtually all the enzyme activity, is collected by centrifugation at 12,000X g. for 10 minutes. It is dissolved in 12 ml. of standard buffer (0.01 M tris buffer, pH 7.4; 0.005 M MgCl 0.0005 M mercaptoethanol), adjusted to 0.4 M (NH SO and allowed to stand overnight at 0 C. This period of waiting is important for the subsequent fractionation since complete disaggregation was found to be essential for acceptable separation of the replicase from transcriptase (transcriptase is the transcribing enzyme which employs DNA as a template to synthesize complementary RNA and is also known as DNA-dependent RNA- polymerase). The extract is diluted with 24 ml. of standard buffer, and after 20 minutes, is centrifuged at for 20 minutes and for each 40 m1. of supernatant are added 12 ml. of a 0.5% solution of protamine sulfate. The precipitate which forms contains virtually all of the DNA- dependent RNA-polymerase along with an RNA-independent RNA-polymerizing activity. The RNA replicase remains in the supernatant and begins to show good dependence on added RNA. (This is one of the critical steps in the fractionation and any variation in host, medium, time or temperature of infection modifies the amount of protamine required to achieve separation. It is often safer to titrate small aliquots and determine the amount of protamine needed by appropriate assays.) After 10 minutes the precipitate is removed by centrifugation at for 10 minutes. To the resulting supernate is added an equal volume of saturated ammonium sulfate (saturated at C. and adjusted to pH 7.0 with ammonium hydroxide). After 10 minutes at 0 C. the precipitate is collected by centrifugation at 12,000 g. for 10 minutes and dissolved in 4 ml. of standard buffer containing 0.4 M ammonium sulfate. The resulting solution is then dialyzed against one liter of standard buffer for 1.5 hours. The dialyzed fraction is adjusted to 0.05 M ammonium sulfate with standard buffer and passed through a DEAE-cellulose column (1.2 10 cm.) which is washed with 100 ml. of standard buffer just prior to use. After loading the protein, the column is washed with 40 ml. of standard buffer containing 0.12 M NaCl which removes protamine, a poly-A synthetase [J. Biol. Chem., 237, 37863793 (1962)] and residual K-dependent ribonuclease. The enzyme is then eluted with 35 ml. of standard buffer containing 0.20 M NaCl. To fractions possessing enzyme activity, saturated ammonium sulfate is added to make the final solution 10% saturation. At this stage, the enzyme preparation has an OD /OD ratio of 1.35 and usually contains 1 mg. of protein per ml. Under the ionic conditions specified, no loss in activity is observed over a month of storage at 0 C.
(E) Centrifugation in CsCl One ml. of 10% ammonium sulfate in standard buffer (0.1 M tris, pH 7.4; .005 M MgCl .0005 M 2-mercapt0- ethanol) containing 10-15 mg. of post-DEAE enzyme protein is layered over 4 ml. of CsCl solution adjusted to a density of 1.40 gm./cm. The tube is centrifuged at 0 C. for 24 hours in the "Spinco SW-39 rotor at 39,000 r.p.m. After centrifugation, the tube is pierced through the side immediately below the visible protein band, and the fractions collected through a 20 gauge hypodermic needle. The needle is bent in a right angle to permit insertion with the long arm up, following which the latter is rotated through 180 to permit the contents to emerge. The lower portion of the tube is then removed by piercing the bottom and collecting fractions. In this manner, contamination of the protein band by the virus band is minimal due to the significant differences in the densities of the two materials. Fractions are then diluted to 1 ml. with 10% ammonium sulfate in standard buffer for optical density measurements and enzyme assays.
(F) Sedimentation through sucrose gradients The peak tubes from the enzyme regions of CsCl gradients are pooled and precipitated from 50% saturated ammonium sulfate. The precipitate is dispersed in standard buffer to a protein concentration of 2530 mg./ml. and the suspension dialyzed for 2 hours against 250 ml. standard buffer at 0 C. Between 0.2 and 0.3 ml. of the dialyzed protein solution is layered onto a 5.4 ml. 5% to 20% linear gradient of sucrose dissolved in standard bulfer. When a reference protein is desired, 100 of catalase is included in each 0.2 ml. of protein solution. Gradients are centrifuged at 0 C. for 12-15 hours at 39,000 r.p.m. in the Spinco SW-39 rotor. Again, to avoid contamination with pelleted virus particles and other materials, the enzyme region is collected from the side as described for CsCl centrifugation. Aliquots are removed from each sample for 0D. measurements and enzyme assays. To avoid dilution inactivation, tubes containing replicase activity are not diluted; aliquots are removed for dilution and OD. determination. Peak activity tubes are pooled and are used Without further treatment. It should be noted that to avoid drastic loss of activity, the purification through sucrose gradients must be done with adequate amounts of protein, not less than 4 mg. per tube.
Purification procedures preliminary to centrifugal purification have been utilized. These procedures involve techniques such as ammonium sulfate fractionation, adsorption and elution from alumina C7 and DEAE cellulose, chromotography on hydroxylapatite gel, isoelectric precipitation, and electrophoresis.
(G) Assay for infectious RNA Protoplasts are prepared by a modification of the method of Guthrie and Sinsheimer [Biochem. Biophys. Acta, 72, 290 (1963)] and Strauss [1. Med. Biol, 10, 442 (1963)]. E. coli K12W6, supplied by Dr. R. L. Sinsheimer of California Institute of Technology, is grown on 50 ml. 3 D medium [1. Biol. Chem, 205, 291 (1953)] to a cell density of 5 1-0 /ml., and harvested by centrifugation at 3,000 r.p.m. for ten minutes at room temperature. The cell pellet is thoroughly drained, and is then dispersed in .35 ml. of 1.5 M sucrose; .17 ml. bovine serum albumin (Armour, 30%); .04 ml. 2 mg./ml. lysozyme (Armour) dissolved in 0.25 M tris, pH 8.1. The resulting suspension is allowed to stand for two minutes at room temperature, during which time any remaining pellet is thoroughly resuspended, then .05 ml. of 4% EDTA (neutralized to pH 7.4 with NaOH) are added. After 30 seconds further, 10 ml. of PAM (1% casamino acids, Difco; 1% nutrient broth, Difco; .1% glucose; 10% sucrose; .2% MgSO the latter added after autoclaving) are added.
Protoplast preparations are stored at 2 C., and used up to five days after preparation. Efficiency of infection by viral RNA reaches a maximum in two or three days after preparation of the protoplasts and remains stable for at least five days. Routine plating efiiciencies achieved are in the range 10* to 10 infectious units per strand of input viral RNA. In assaying replicate products for infectivity, aliquots from reaction mixtures are diluted in .01 M tris, pH 7.4; .005 M MgCl to an RNA concentration of 0.2 to 0.8 v/ml. (as defined by counts incorporated during synthesis). 0.2 ml. of the diluted reaction mixture are brought to 35 C., and 0.2 ml. of protoplast suspension at room temperature are added. After 30 seconds at 35 C., the mixture is diluted five-fold with PAM broth, incubated at 35 C. for a further 10 minutes and then plated. The indicator organism is E. coli K38, supplied by Dr. N. Zinder of Rockefeller University. Plates are incubated at 34 C.
After the initial fractionation on DEAE cellulose [at page 581 of Proc. Natl. Acad. Sci, U.S., 54, 579 (196 5)], replicase preparations contain from 10 to 10 plaqueforming particles per mg. of protein. To permit direct assay of reaction mixtures for infectious RNA requires reduction of virus particle content by factors of 10 Initial separation of protein and virus by density is achieved by equilibrium centrifugation in CsCl density gradients. Residual virus particles are removed by sedimentation in sucrose gradients.
In the accompanying drawings (FIGURES 1-7), data are shown for replicase preparations which have gone through the CsCl and sucrose centrifugation steps only: FIGURE 1 shows the results of banding of enzyme protein in a CsCl gradient; FIGURE 2 shows the comparison of primer response by the replicase before and after CsCl purification; FIGURE 3 shows a comparison of autocatalytic synthesis by the replicase before and after CsCl purification; FIGURE 4 shows the behavior of the replicase during sedimentation in sucrose gradients; FIGURE 5 shows the resolution of replicase and transcriptase in sucrose gradients; FIGURE 6 shows the sedimentation analysis of extensive synthesis by the replicase purified through CsCl and sucrose; and FIGURE 7 shows the synthesis of RNA and infectious units by enzymes purified through CsCl and sucrose centrifugation.
Pycnographic purification.The results of banding the enzyme protein preparation obtained after the first DEAE chromatography step in a CsCl gradient are presented in FIGURE 1. In order to obtain these results, refractive indices were read, and then samples were diluted to 1 ml., and 20A of each fraction were examined for enzyme activity in the standard reaction mixture; incubation conditions were 30 C. for 30 minutes. Virus particles are found at a density corresponding to 1.41 gm./cm. Replicase bands at 1.26 gm./cm. somewhat less dense than bulk protein, and is therefore easily separated from the virus band. Approximately 10* of the original phage contamination is retained in the bulk protein peak, and repeated banding of the protein in CsCl failed to reduce the contamination substantially. Presumably the protein precipitate formed in the high concentrations of CsCl acts as an ion exchange surface, with the residual virus contamination representing the adsorption capacity of the precipitate.
After purification in CsCl, the requirement for and response to added Qfi-RNA is retained. However, as is shown in the saturation curves of FIGURE 2, there is a substantial increase in the observed activity of the enzyme following pycnography. In order to obtain these curves, standard reaction mixture contained 50 g. of enzyme protein; specific activity of triphosphate was 3x10 c.p.m./0.2 am; incubation conditions were 30 C. for 30 minutes. Since equivalent concentrations of RNA are required to saturate the enzyme, the stimulation observed after CsCl would seem to reflect a more rapid read-out of the template. This implication is borne out by an examination of exponential synthesis during template limitation.
FIGURE 3 is a comparison of autocatalytie synthesis by replicase before and after CsCl purification; the doubling time of RNA is three times that catalyzed by enzyme which was not further purified. In order to obtain this comparison, reactions containing 50 g. protein and .15 g. Qfi-RNA in each standard reaction mixture were carried out at 30 C. and aliquots were withdrawn at the indicated time intervals; the specific activity of triphosphates was such that 3,300 c.p.m. corresponded to 1 ,ug. RNA product in each 0.25 ml.
Sedimentation purificalion.Protein preparations obtained after banding in CsCl still contain too many virus particles to permit direct assays of reaction products for infectivity. The residual contamination is lowered to acceptable levels by sedimentation of the CsCl-purified enzyme protein through linear gradients of sucrose. FIG- URE 4 shows the sedimentation profile of replicase activity, with catalase included as a reference. After 12 hours of centrifugation, infectious virus particles are found as a pellet in the tube. It will be noted that replicase activity sediments as a single peak. Employing catalase (molecular weight, 2.5 10 as a standard, the molecular weight of replicase may be estimated at Table 1 summarizes the relevant properties of the replicase preparation at each stage of the purification. More specifically, the recoveries of replicase activity, viral contamination, and relative optical density measurements during a purification are given in Table 1. Quite often, O.D. "/O.D. ratios of post-DEAE enzyme preparations are as high as 1.0. After CsCl and sucrose, however, this is invariably lowered to 0.60, indicating effectiveness of the technique. Also, activity recoveries from CsCl often exceed 100%.
TABLE 1.PROPERTIES OF REPLICASE AI DIFFERENT STAGES OF PURIFICATION The final residue of phage contamination is such that only 1 to 10 plaque-forming units are introduced into a standard reaction mixture, a level which would not be detected by our usual sampling procedure.
Synthesis routinely involves the appearance of 0.2 to 20 of new RNA, corresponding to 10 to 10 infectious units per reaction mixture under our assay conditions. The levels of mature phage contamination are clearly far below detectability.
Occasionally some transcriptase (DNA-dependent RNA-polymerase) contaminates the replicase peak eluted from the DEAE column. Rechromatography is recommended when this occurs. However, as FIGURE 5 shows, sedimentation through sucrose gradients can be used to separate replicase from transcrintase. This procedure is also extremely effective in eliminating any remaining traces of RNAase, which remain at the top of the gradient. In obtaining the data for FIGURE 5, centrifugation, collection and assays for replicase are carried out as described above; and in assaying for DNA-dependent activity, 10 g. calf thymus DNA were substituted for QB-RNA in the standard reaction mixture; and from relative sedimentation rates of the two activities, the molecular weight of the transcriptase was estimated at 3 X 10 Synthesis of infectious nucleic acid.We have already shown that the response of replicase to template is retained throughout the purification. Further, FIGURE 6 establishes that the product of the purified enzyme possesses a sedimentation pattern similar to Qfi-RNA. In obtaining the sedimentation analysis of FIGURE 6, the standard reaction mixture contains 50 g. of purified enzyme protein and 0.2 g. of Qfi-RNA; the reaction is continued for 40 minutes at 35 C. during which a 30- fold synthesis occurs. To 0.1 ml. of the reaction mixture were added 0.01 ml. of 20% sodiumdodecylsulphate, 0.005 ml. H QB-RNA, and 0.20 ml. .01 M tris, pH 7.4; .005 M MgCl it was then layered onto a linear gradient of 2.5% to 15% sucrose in 0.01 M tris, pH 7.4; .005 M MgCl .1 M NaCl, and centrifuged at 25,000 r.p.m. for 12 hours at 10 C. in the Spinco SW-25 rotor.
As shown in Proc. Natl Acad. Sci., U.S., 54, 919 (1965), an enzyme purified through the first DEAE stage provides convincing evidence that RNA synthesized by replicase possesses the biological information characteristic of Q5. In these experiments, the product of a reaction initiated with Qfi-R-NA is serially diluted in successive reactions until the original Qfi-RNA is reduced to an insignificant level. The final tube contains new radioactive RNA which is fully as competent biologically as the viral RNA used to start the reaction in the first tube.
We now offer a similar demonstration showing that replicase further purified through CsCl and sucrose gradients retains unimpaired the ability to generate biologically active copies.
The reaction is initiated at less than primer saturation, and synthesis is allowed to proceed. After a suitable interval, 2 5 of this reaction is used to initiate a second one. Again, after permitting adequate synthesis, the same aliquot of the second tube is employed as the template for a third reaction, and so on for 10 transfers. FIGURE 7 shows the synthesis of RNA and infectious units by purified enzymes. Since the reaction in the first tube is started with 6 10 strands (0.17) of Qfl-RNA and each transfer involves a dilution, the combination of the initiating RNA to the infectious centers measured in the fourth tube is below the level of detectability, and this tube contains 2.4)(10 infectious units. Finally, the eleventh reaction contains less than one strand of the initial primer and at the same time shows 2.5)(10 infectious units as determined by plaques formed in the protoplast assay. Clearly, the replicase purified by pycnography and sedimentation has retained its ability to produce biologically competent replicas.
In obtaining the data for FIGURE 7, eleven reaction mixtures of .125 ml., each containing 22 g. of enzyme purified through CsCl and sucrose centrifugation and the other components of the standard reaction mixture were prepared. Specific activity of P UTP was such that 8,000 c.p.m. signified 1 ,ug. RNA product. To the first tube were added 0.1 g. Q/8-RNA, and the reaction was allowed to proceed for 25 minutes at 35 C., whereupon .02 ml. were withdrawn for counting and .01 ml. used as template for the second reaction. The first tube was then frozen and stored at 70 C. The second reaction product was used to initiate the third and so on. A second series of transfers were carried out in a manner identical to that described, save that no initial RNA template was added to the first reaction mixture. Aliquots of all the reaction mixtures were directly assayed for infectious units as described above. In the case of the control transfer series, samples were diluted A in TM and then mixed with protoplasts. All other samples were adjusted to 0.2 to 0.8 pg. RNA product/ml. before mixing with protoplasts.
These methods, detailed above, for purification of QB replicase yield preparations sufiiciently free of virus particles to permit direct assay of the RNA product for biological activity.
We claim:
1. The method of synthesizing in vitro a biologically active intact ribonucleic acid comprising the steps of providing an activated system capable of synthesizing in vitro biologically active ribonucleic acid for prolonged or extensive periods, which system includes biologically active intact ribonucleic acid; the specific replicase for said ribonucleic acid which will recognize the intact ribonucleic acid of its origin and which is free of detectable destructive contaminants and substantially free of vital infectivity; the nucleotide base components adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphtae; and divalent magnesium ions as an activating cofactor; and allowing said system to incubate.
2. The method of claim 1 wherein said ribonucleic acid is a viral ribonucleic acid.
3. The method of claim 1 wherein said nucleotide base components are isotopically labeled.
4. The method of claim 1 wherein said synthesized ribonucleic acid is recovered from said system.
5. The method of synthesizing in vitro biologically active, intact ribonucleic acid involving copying the sequence of bases thereof, comprising the steps of providing an activated system in vitro which includes biologically active, homologous, intact ribonucleic acid; the specific replicase for said ribonucleic acid which will recognize the intact ribonucleic acid of its origin and which is free of detectable degrading enzymes, and inhibitors, and substantially free of viral infectivity; the nucleotide base components adenosine triphosphate, guanosine triphosphte, cytidine triphosphate, and uridine triphosphate; and divalent magnesium ions as an activating cofactor; and allowing said system to incubate.
6. The method of claim 5 wherein said synthesized ribonucleic acid is recovered from said system.
7. The method of reproducing biologically active, intact ribonucleic acid in vitro, which method comprises: providing an in vitro, enzymatic, self-duplicating system hav-l ing (a) biologically active, homologous, intact, ribonucleic acid template, (b) the specific selective active homologous replicase for said ribonucleic acid that has a selective preference for the homologous template and which will recognize the intact ribonucleic acid of its origin, said replicase being effectively free of detectable nuclease activity and destructive enzymological activity, and substantially free of viral infectivity, (c) the nucleotide base components adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate; and (d) divalent magnesium ions as activating cofactors; allowing said system to incubate and recovering biologically active ribonucleic acid produced from said system.
8. The method of claim 7 wherein said ribonucleic acid is a viral ribonucleic acid.
9. The method of claim 7 wherein said ribonucleic acid is ribonucleic acid of bacteriophage Q 3.
10. The method of claim 7 wherein said ribonucleic acid is ribonucleic acid of bacteriophage MS2.
11. The method of claim 7 wherein said system is significantly free of transcriptase and deoxyribonucleic acid.
12. The method of claim 7 wherein said system includes deoxyribonucleic acid and said replicase includes transcriptase.
13. The method of claim 7 wherein said base compo nents are isotopically labeled.
14. The method of reproducing replicas of intact viral ribonucleic acid free of proteinaceous coating in vitro, which method comprises providing an invitro, enzymatic, self-duplicating system having (a) a biologically active, self-propagating, homologous, intact, viral ribonucleic acid input template free of a protein coating; (b) the active specific replicase for said viral ribonucleic acid that has a selective preference for said template and which will recognize the intact ribonucleic acid of its origin, said replicase being effectively free of detectable nuclease activity and destructive enzymological activity, and substantially free of viral infectivity, (c) the nucleotide base components adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate; and (d) divalent magnesium ions as activating cofactors; and in which the system does not have transcriptase present together with deoxyribonucleic acid; and allowing said system to incubate.
15. The method of claim 14 wherein said replicas are recovered from said system.
16. A replicase for viral RNA, said replicase being effectively free of detectable levels of degrading enzymes and inhibitors, and substantially free of viral infectivity, being able to recognize the intact ribonucleic acid of its origin, and being capable of providing prolonged or extensive synthesis of homologous, intact viral RNA from adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, uridine triphosphate, and magnesium ions.
17. The product of claim 16 wherein the replicase has antigenic properties.
References Cited Proc. Natl. Acad. Sci., vol. 50, pp. 905-911 (1963).
ALVIN E. TANENHOLTZ, Primary Examiner.
US. Cl. X.R.
|Citing Patent
|Filing date
|Publication date
|Jan 14, 1987
|Jan 3, 1989
|President And Fellows Of Harvard College
|T7 DNA polymerase
|Dec 14, 1987
|Jul 17, 1990
|President & Fellows Of Harvard College
|T7 DNA polymerase
|Mar 1, 1990
|Aug 7, 1990
|President And Fellows Of Harvard College
|T7 DNA polymerase
|Apr 16, 1986
|Sep 18, 1990
|The Salk Instute For Biological Studies
|Replicative RNA reporter systems
|Dec 6, 1988
|Feb 19, 1991
|President And Fellows Of Harvard College
|DNA sequencing
|Mar 7, 1990
|Sep 8, 1992
|President & Fellows Of Harvard College
|Method of using T7 DNA polymerase to mutagenize and fill-in DNA
|Sep 4, 1992
|Nov 30, 1993
|President And Fellows Of Harvard College
|Method of using T7 DNA polymerase to label the 3' end of a DNA molecule
|Sep 22, 1992
|Nov 15, 1994
|The Salk Institute For Biological Studies
|Replicative RNA reporter systems
|Apr 29, 1994
|Feb 11, 1997
|The Trustees Of Columbia University In The City Of New York
|Cell-free method for synthesizing a protein
|Feb 14, 1994
|Jun 17, 1997
|President & Fellows Of Harvard College
|Method for sequencing DNA using a T7-type DNA polymerase and short oligonucleotide primers
|Jun 6, 1995
|Feb 16, 1999
|The Trustees Of Columbia University In The City Of New York
|Autocatalytic replication of recombinant RNA
|Sep 13, 2002
|Mar 27, 2003
|Universite De Liege
|Preparation of triphosphosrylated organic compounds optionally marked with phosphorus 32 or phosphorus 33
|U.S. Classification
|435/91.21, 435/194, 435/91.3, 435/192, 435/199
|International Classification
|Cooperative Classification
|European Classification


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By Garold L. Markle
I am frequently asked to describe what differentiates a successful employee development program from a less successful one. In a word, my answer is "coach-ability." If I could do only one thing to maximize the chance that a supervisor's performance improvement advice would be followed by a direct report, I'd focus on helping the direct report understand WHY a positive predisposition toward change might be in the best interest of the direct report, her supervisor and the organization. Fertile soil makes growth much easier.
I recently had an opportunity to observe a company owner give her General Manager four improvement areas on which to concentrate in the coming year. One of those areas for growth was to stand up and take a more visible leadership role in organization-wide meetings. The recommendation was conveyed as part of a coaching session that concluded at 2:00 in the afternoon. At 3:00 there was a holiday meeting with all employees present. The company owner stood up to lead the meeting, as usual, when the GM asked if he could say a few words to the assembly. Speaking from freshly prepared notes, he proceeded to recap the current year, forecast the next and publicly commit to conducting more of these meetings in the coming year. I cannot think of a more commanding way in which to communicate to the owner "Message received!" Now that is what I call coach-ability.
Here's a second example. The Operations Manager of a large services organization stated in her Catalytic Coaching Employee Input Session that she was interested in being considered for the CEO position in the five year or later time horizon. She made this statement wearing blue jeans and a faded polo shirt with a vendor logo prominently featured. The person to whom she was presenting the information was wearing a suit and tie. As you never knew when an important client or benefactor might drop by, this is how he came to work every day.
As part of the coach's effort to "say to the employee what you would say more comfortably about her to someone else," the CEO coach established the theme of "Image Management" as her fourth Area for Improvement. As she later described it, "He actually talked about the giant white elephant in the room." She knew that her personal dress habits were lax but the internal focus of her operations position did not require anything more formal. As part of his counsel, he described to her the need to help the Board of Directors see in her what he already saw — a legitimate candidate for succession. The casual clothing was a potential distraction and created an image of her not in keeping with the position she coveted.
Despite her knowledge of the wardrobe delta, hearing it articulated out loud was a bit challenging. The next morning, however, she came to work in a newly purchased pant suit. She decided to "run for office" and to start doing things in keeping with the role she wanted instead of the role she currently occupied. Message sent. Message received. Another great example of coachability.
Bottom line, if you want to make rapid and significant behavioral change in your direct reports, take the time to insure that the groundwork has been laid for positive reception to your suggested areas for improvement. Teach them WHY it's in everyone's best interest for them to make changes and you, too, will create an atmosphere of coach-ability.
Garold Markle is author of Catalytic Coaching: The End of the Performance Review and No More Performance Evaluations! Gary is also founder and CEO of Energage, Inc. For more of his teachings go to Energage.com.
This article was first published in Catalytic Connection in April 2008.