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NORWAY: Duty free sales threatened by tax proposals
By just-drinks.com editorial team | 11 July 2007
The Norwegian government's Committee on Tax Reform (CTR) has proposed a new tax initiative that would effectively lead to the revoking of licences that currently allow the sale of duty free alcohol at the country's airports and on passenger ferries operating in Norwegian territorial waters, Jens Stoltenberg, Norway's Prime Minister has told the national parliament.
"We are awaiting a report from the committee quite soon. This will be an important signpost for future reforms within the area of alcohol sale in Norway. The continuance of duty free sales in their present format will also be looked at," said Stoltenberg.
The CTR was to have presented its initial report on possible reforms at the end of June. This report, which is also expected to propose a 10% increase in taxes on spirits and beer products, is not now expected until September, or October.
The brewing industry has reacted critically to the CTR's proposals.
"The only beneficiary of a decision to raise taxes on alcohol will be foreign countries and especially Sweden, as a tax hike would produce a strong increase in cross border imports of alcohol. This reform may sound the death of the brewery industry in Norway," said Lars Midtgaard, CEO of Borg Breweries.
Sectors: Beer & cider, Spirits, Wine
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Master in Economic Geology
Master in Economic Geology
The Master's Programme in Economic Geology is offered by Oulu Mining School, with extensive international linkages.
The programme will be taught entirely in English and comprises 22 modules, including courses in magmatic, hydrothermal and sedimentary ore deposits, structural geology for economic geologists, mining geology, geometallurgy, sampling methods, GIS applications, as well as environmental geochemistry.
Employment Opportunities for Graduates
Northern Finland is currently experiencing an unparalleled mining boom, making the area one of the exploration and mining powerhouses of Europe. The minerals sector may become one of the key foundations of the Finnish economy as commodity prices are bound to continue their rise on the long term and demand from China and other emerging economies is predicted to grow. These factors ensure employment opportunities for graduates as well as funding opportunities for future research projects.
Programme Outline
The Master's degree programme comprises 120 ECTS, including a 35 ECTS Master's thesis, and between 50 and 85 ECTS of advanced courses in Geology and up to 35 ECTS from related disciplines to be chosen freely.
Programme Specific Academic Requirements
The programme is open for applications from students with a Bachelor's degree in geology, or other sciences, as long as students have sufficient background in geology (60 ECTS credits).
Students who are in their last term/semester of Bachelor's studies can apply to study for a Master's degree. In the case that the applicant is accepted for study, the applicant has to submit his/her Bachelor's degree documentation by the following deadlines.
Selection Process
After preliminary selection, the applicants will be interviewed by telephone, during which the educational background of the applicants, their motivation for study, and their knowledge of English are evaluated.
Detailed Selection Criteria
Selection is mainly based on academic performance and relevant experience, e.g., in industry or geological survey organizations.
Student Selection
The names of the selected candidates are announced in April 2015.
This school offers programs in:
- English

subset: commoncrawl


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Business ManagementMr. Steve Nero, Owner Mrs. Ideal Gandarilla, Customer Service Manager Mr. Mike Kampff, Vice President
Contact Information
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Party Supplies - Renting Event Planners Childrens & Family Entertainment


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Tag Archives: J.T. Tartacoff
Morning kickoff: Rutgers aftermath
***Without hyping Teddy Bridgewater up too much, which at this point I don't know if that's possible, I think his performance can be deemed the start of his 2013 Heisman Trophy campaign.
His actual numbers – 20 of 28 attempts for Continue reading
Posted in Uncategorized Tagged Calvin Pryor, Charlie Strong, Corvin Lamb, D.C. Jefferson, J.T. Tartacoff, Jamon Brown, Kenny Carter, Louisville football, Preston Brown, Teddy Bridgewater, Tom Jurich Comments Off


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Robert Abbey has been designing and manufacturing fine lighting since 1946. They offer a diverse collection--wall swingers, chandeliers, floor lamps and more--in myriad styles, from tradition to neoclassical to groovy. Collaborating with acclaimed designers Jonathan Adler, Rico Espinet and David Easton, Robert Abbey creates impeccable lighting that is perfect for modern everyday living.
The Saturnia Wall Sconce is available with the following:
- Cylinder-shaped, transparent outer fabric shade
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- Metal body
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- Installation Type: Plug In with Pin-Up Kit Included
- UL Listed
- Made In China
Inner Shade Color: Ascot White
Finish: Deep Patina Bronze with Bronze, Stainless Steel with Silver Transparent
- One 60 Watt (672 Lumens) 120 Volt E12 Candelabra Base Incandescent Lamp(s) (Not Included)
Compare Brightness:
LumensThe amount of light that comes from a bulb, which has commonly been measured in watts. The more lumens, the brighter the bulb.
WattageA measurement of the power delivered to a component of an electric circuit (allowing a one-ampere current to flow through the component under the pressure of 1 volt.)
Bulb ShapeBulb shapes are denoted with a letter, which describes the shape, and a number, which indicates the size. The number indicates the diameter of the light bulb at its widest part in eighths of an inch.
BaseThe part of the bulb that connects to the fixture and its power supply. Bulb bases are denoted with the letter E, and a number which indicates the diameter of the bulb base at its widest part in millimeters.
VoltageIndicates how much voltage a bulb can accept via the fixture it's installed in.
Light Color TemperatureThe relative color from a light source, measured on the Kelvin temperature scale. Warm light is lower in color temperature (2700-3000 K) and cool, blue light is higher (5000 K).
Average Rated LifeThe average amount time that a bulb is expected to last.
CRICRI = color rendering index. This number, which can go from 0 to 100, measures the ability of a light or bulb accurately renders colors. The higher the number, the better, with most quality bulbs these days measuring at least in the 80s.
- Fixture Width 8", Height 12", Depth 10.25":
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April 24, 2009 — Earl Mark, associate professor and chief technology officer in the University of Virginia School of Architecture, has been named Educator of the Year in the 2009 "Be Inspired" awards program, sponsored by software developer Bentley Systems.
Following a nomination process, entrants submitted a portfolio, which was subjected to competitive review by an independent jury of academics and practitioners from the disciplines of architecture and engineering. The field of nominees was drawn from higher educational institutions internationally. The award will be conferred at a banquet to be held in September.
For more information visit the awards Web site.


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Worthy Objects: Hassan Rahim Talks Shop with Use All Five
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Photo Essay: Let's Open, Like, 1700 Baseball Cards
Part I
At the risk of losing a considerable amount of 'Net cred, I feel as if I need to make a confession; I haven't bought a baseball card in about 20 years. There are lots of reasons for this, the main reason being that my friends stopped doing it first. As an adolescent who strove to be accepted, I had no interest in doing anything which my group of friends had no interest. I considered this to be, at the time, a form of self-betterment. I wanted nothing more than to hang on to the small group of friends I had, and therefor the "childish" activity of opening, sorting, and saving baseball cards was done away with.
I am now older and wiser. I understand that having hobbies and interests that are my own within my group of peers is acceptable and healthy. I embrace it. I also am on a different kick of self-improvement, related directly to baseball. As a young baseball fan, I was really only interested in the best players. I did not pay heed to the men I deemed unworthy of my attention. Superstars were it for me. This has led to a considerable knowledge gap when it comes to players of the late '80s and early '90s. Too many players have slipped through the cracks – players that weren't very good or perhaps were good, but I failed to recognize their contributions since I had yet to fully understand player value.
This all being said, I decided to resurrect my baseball card hobby as both a pleasurable exercise, and as a vehicle for reacquainting myself with forgotten players. I therefor decided to purchase a collection of baseball cards from that time. Deploying the services of eBay and my local team's fan festival, I acquired approximately 1700 baseball cards for what I consider to be a very reasonable price.
I set aside a few cards that caught my attention, and have decided to share them with you, fair NotGraphs reader. I hope you'll join me in this trip down memory lane. I'm sure many of you remember these cards and players, but, this is about me. It always is. Shall we?
That's all for today. More to come tomorrow. Thanks for taking this trip with me.
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Tag: finances
Protect Yourself and Your Family Against Identity Theft
How the Financial Crisis Affects Our Health
Dear Mr. Dad: The ongoing financial crisis has been really hard on my family. We haven't had a...Read More
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Dear Mr. Dad: Right now, I work fulltime and my wife is home with our twin preschoolers. However,...Read More
Daddy, why can't you just get money out of one of those machines?
When my first child, who's now 22, was less than a year old, I took her to an FAO Schwartz store in New York, thinking that she'd have a ball with all the toys and stuffies. But no matter what I pulled off the shelf,...Read More
What parents need to know about money
No one ever wants to talk about money issues because they're afraid it'll lead to a fight. Well, guess what? Not talking about it could lead to a financial meltdown that could destroy your relationship. Fight now vs....Read More
There's More to Being a Dad Than Pulling out Your Wallet
Dear Mr. Dad: I have a 13-year old daughter. I was never married to her mother. But recently the mom got married to someone else, had another child, and moved away, taking my daughter with her. Do I still have to pay child...Read More


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Rent Principles of Macroeconomics 4th edition today, or search our site for other textbooks by John B. Taylor. Every textbook comes with a 21-day "Any Reason" guarantee. Published by McGraw-Hill.
Need help ASAP? We have you covered with 24/7 instant online tutoring. Connect with one of our Macroeconomics tutors now.


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Red Carpet News
Back On? Idris Elba Walks the Red Carpet with Naiyana Garth 3 Months After Split Rumors
Originally posted 05/08/2016 05:55PM
What breakup?
The Stars Turn Out for the BAFTA TV Awards Red Carpet: See Justin Timberlake, Anna Kendrick and More
Originally posted 05/08/2016 03:30PM
Television award season may be over in the United States, but across the pond the red carpet is still being rolled out.
Robert Downey Jr. Attends Captain America: Civil War Premiere With Wife Susan
Originally posted 04/13/2016 01:30AM
Robert Downey Jr. hit the red carpet with his leading lady, wife Susan, on Tuesday when the pair attended the premiere of Captain America: Civil War in Los Angeles.
Sarah Paulson Reveals What Fans Should Expect From the Finale of The People v. O.J. Simpson: American Crime Story
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Kim Kardashian West Hits Back at Body Shamers After Sharing Nude Photos: 'I Am Empowered by My Sexuality'
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Kim Kardashian West is speaking out following a backlash that ensued when she shared a nude photos of herself on social media.
Inside Leo's Big Night Out: DiCaprio Parties with Revenant Costars After BAFTAs Sweep
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Leonardo DiCaprio Says the Cold While Filming The Revenant Was Worse Than the Icy Waters in Titanic
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'Mom, Happy Birthday!' Leonardo DiCaprio Wins Best Actor BAFTA for The Revenant
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BAFTAs 2016: The Revenant Wins Best Picture
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Dell AC411 2.1 Wireless Speaker System - Affordably Functional
The Dell AC411 is not just your run of the mill affordable 2.1-channel speaker system. It even supports wireless playback thanks to its Bluetooth capabilities. But will its 33W sound output help it make the grade? Details in our review!
Dell UltraSharp U2713HM - Premium Price for Premium Picture Quality
The latest 27-inch model to the UltraSharp desktop monitor family features an IPS panel with LED backlighting. In a matte-black finish and silver highlights, the Dell U2713HM lets you adjust height, tilt, as well as swivel. Read on to find out how we rank this one.
Hands-on: Dell XPS 12
With Windows 8's Modern UI designed with touch in mind, manufacturers are steadily introducing more and more hardware with touch-enabled screens. The latest is Dell's XPS 12, which uses a swiveling screen form factor to transform between tablet and notebook. Find out how well it works inside:
Dell Unveils XPS Duo 12, XPS 10 Tablet/Notebook Hybrids & XPS One 27 AIO PC at IFA 2012
Dell has announced three new Windows RT and Windows 8 devices: the XPS Duo 12 and XPS 10 tablet/notebook hybrids, and the XPS One 27 AIO PC. The star of the bunch is the XPS Duo 12 which sports a rotating screen.
Meet Dell's New Ultrabooks - The XPS 14 and Inspiron 14z
Dell added two new models to their Ultrabook range this week. Joining last year's XPS 13 is its bigger brother, the XPS 14 and the student-targeted Inspiron 14z. Dell brought both models down to the HardwareZone offices for us to take a closer look. Find out more inside!
Dell XPS 13 - Fast, Compact and Portable
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Google links digital ads and offline sales
The search giant today rolled out new ways for marketers to understand the in-store impact of their ads.
Google links digital ads and offline sales
The search giant today rolled out new ways for marketers to understand the in-store impact of their ads.
Online-only home furnishings retailer Dot and Bo shuts down
Dot and Bo, in business for three years, focused its furniture and decor items toward millennial customers.
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Sponsored by 2013 Social Media 300
The social network sets its own value at $77 to $96 billion.
Facebook Inc. today announced it plans to price its shares for its initial public offering between $28 and $35. That price range would value the company at between $77 billion to $96 billion, which far outpaces the $23 billion valuation Google Inc. received when it went public in 2004.
The social network announced the valuation in a filing with the U.S. Securities and Exchange commission.
The filing notes that Facebook plans to sell up to 337.4 million shares, roughly half of which are being sold by founders, employees and investors. With Facebook selling 180 million shares, the company can raise as much as $6.3 billion in working capital via the IPO.
Advertising accounts for the vast majority of Facebook's revenue. In 2009, 2010, and 2011, ads on the social network accounted for 98%, 95%, and 85%, respectively, of revenue. However, investors may be cautious as the social network recently reported its first quarter earnings decreased 12% year over year, to $205 million from $233 million. While that was due in part to the social network's quarterly expenses rising to $677 million from $343 million a year earlier, it could give some investors pause, particularly after another high-profile Internet company , Groupon, has seen its value plummet because it failed to generate profits after its November IPO.
Marketers advertise on Facebook because consumers share a wealth of data on the social network, which, in turn, advertisers can use to target consumers with ads. The amount of data Facebook has is rapidly growing, as Facebook's 901 million members add 3.2 billion Likes or comments a day, an average of 3.6 interactions per user per day.
Images, tags and mobile performance are among the key areas to focus on in the ...
Posted 09/23/16
Many e-retailers focus on conversion rate, but here are four less-obvious ways to analyze your ...
Posted 09/23/16
Online retail is hard, grocery retail is really hard, so online grocery is, of course, really, really hard."


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[Verse: Markita]
Somebody made me an offer and I accepted
Forever in debted to weapon
Love, honor, and respected
But his image was different than what his character reflected
Word is he's seven-thirty his hands never dirty
All I know this so called psycho never hurt me
Touch me down to tips, shopping sprees on Fifth
With enough dough that could be carry out on forklifts
Earnt his trust, together till we turn to dust
Until his theory disintegrated and burnt to crust
What? What?
Death to a soft heart
That's what I vow to you
The vows I vow to you
Amazing how it's true
Talk goes on our phone
Just to remind you
That I'm behind you
The FEDS won't find you
The 5th song from Kelis' debut album, 'Kaleidoscope'.


text:
|Publication number
|Publication type
|Application number
|Publication date
|Sep 1, 1998
|Filing date
|Jun 1, 1995
|Priority date
|May 25, 1984
|Fee status
|Publication number
|William Alan Haseltine, Craig A. Rosen, Joseph Gerald Sodroski, Wei Chun Goh
|Original Assignee
|Dana Farber Cancer Institute
|Export Citation
|BiBTeX, EndNote, RefMan
|Non-Patent Citations (41), Referenced by (3), Classifications (15), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a divisional of application Ser. No. 08/213,368, filed on Mar. 14, 1994, now abandoned, which is a continuation of No. 07/869,053, filed Apr. 14, 1992, now abandoned, which is a continuation of No. 07/604,607, filed Oct. 26, 1990, now abandoned, which is a divisional of No. 06/806,263, filed Dec. 6, 1985, now Pat. No. 4,981,790, which is a continuation-in-part of No. 06/614,297, filed May 25, 1984, now Pat. No. 4,738,922.
The present invention is directed to the establishment of cell lines that stably express the tatIII gene, and the use of the gene product. More particularly a protein and a polypeptide are described which can dramatically increase the rate of expression of a heterologous gene product.
Much effort has been spent over the years in attempting to understand the mode of action of viruses, particularly that of retroviruses. Questions for which answers have been sought include the reasons that certain of these viruses preferentially infect and/or replicate in certain types of cells as opposed to other types of cells.
The acquired immune deficiency disease (AIDS), and AIDS-related complex are the subjects of intensive scientific research and public concern. Human T-Cell leukemia virus III (HTLV-III)/LAV is the etiological agent of the acquired immune deficiency disease, AIDS-related complex and other virus-related disorders including degeneration of the central nervous system, lymphoid interstital pneumonitis (LIP) an increased incidence of Kaposi's sarcoma, B-cell lymphoma of a Burkitt's type, Hodgkin's lymphoma and thrombocytopenic purpera, collectively called HTLV-III/LAV related disorders (1) F. Barre'-Sinoussi et al., Science 200:868 (1983): R. C. Gallo et al., ibid., 224:500 (1984); J. Schupback et al., ibid.: 503; M. G. Sarngadharan et al., ibid.: 506; J. A. Levy et al., ibid.: 225,840 (1984); D. Klatzmann et al., Nature (London) 313:767 (1984); M. Gottlieb et al., New England J. Med. 305:1425 (1981); H. Masur et al., ibid.: 1431; F. Siegal et al., ibid.: 1439; H. Lane et al., ibid.: 309,453 (1983); J. Ziegler et al., ibid.: 311,565 (1984); G. Shaw et al., Science 227:177 (1985). (2) D. Klatzman et al., Science 225:54 (1984); M. Seligman et al., New England J. Med. 311:1286 (1984).! AIDS is clinically typified by depletion of T-Cells of the T4+(helper) subset, a phenomena reflected by cytotoxicity of the virus for T4+cells in vitro (2). Large scale production of the virus was made possible by the development of T4+ cell lines that were susceptible to virus infection but that were partially resistant to its cytopathic effects (3) M. Popovic et al., Science 224:497 (1984).!
We have previously discovered genes in HTLV-I, HTLV-II, and HTLV-III, which code for proteins resulting in trans-activation of their respective viruses. (See PCT/U.S. No. 85/00985 filed May 24, 1985 which is incorporated herein by reference). These genes have been previously referred to as x-lor genes as well as luk genes, but have been collectively re-named tat genes using the conventional method of naming genes by function.
Being able to establish cell lines that stably express the tatIII gene without the possibility of virus spread provides an opportunity to study the biochemical events associated with tatIII gene expression in vivo, as well as obtaining large scale quantities of the tatIII gene products, and/or a desired heterologous gene product.
We have now discovered a method for producing cell lines that will stably express the tatIII gene in vitro using defective HTLV-III/LAV virus, thus eliminating the possibility of virus spread in the absence of helper virus. These tatIII cell lines can be used to express a large quantity of a desired gene product.
In addition, we have discovered a protein of approximately 14,000 daltons consisting of 86 amino acids, which is the functional tatIII protein. A polypeptide weighing only approximately 12,000 daltons molecular weight, containing the first 69 amino acids of the tatIII protein will also provide activity similar to that of the tatIII protein.
The tatIII cell line and/or the gene product can be used in the production of large quantities of a desired product from a chosen tissue by using a modified LTR vector containing a desired heterologous gene product.
The tatIII cell lines of the present invention can also be used for assay purposes. These cells can be used to detect the presence of HTLV-III/LAV virus in individuals more easily and with greater sensitivity than current diagnostic methods.
Additionally, these cell lines can be used to screen for compounds that inhibit the trans-activation of the tatIII gene product. This method includes the steps of:
(a) transfecting a tatIII cell line with a vector containing a gene that expresses a selectable marker and whose expression is under the control of art HTLV-III LTR;
(b) transfecting the same type of tatIII cell line as in step (a) with the selectable gene chosen in step (a) but under the control of a different regulatory sequence;
(c) thereafter adding a preselected compound to each of the cell lines in increasing concentrations; and
(d) measuring the expression of the selectable gene product to determine whether the compound effects the tatIII function without being toxic to the cell.
FIG. 1 illustrates a method according to the present invention of establishing a cell line containing the tatIII gene.
FIGS. 2A and 2B illustrates the dramatic difference in the CAT activity seen in infected vs. uninfected cells.
FIG. 3 illustrates the plasmid used to assess the trans-activation.
FIG. 4 illustrates the structure of HTLV-III/LAV deletion mutants that retain trans-activating activity.
FIG. 5 shows the construction of a tatIII fusion product in a bacterial open reading frame infector.
FIGS. 6A-E illustrates the expression of fusion proteins that contain the tatIII protein.
FIGS. 7A and B represents the nucleic acid sequence of the tatIII gene and the predicted amino acid sequence. FIG. 7A shows a schematic of the HIV genome with the tat nucleic acid shown underneath and the beginning of the nucleic acid sequence of the tat gene with the predicted amino acid sequence through base No. 5406. FIG. 7B shows the remainder of the nucleic acid and nucleic acid sequences for the tat gene.
FIG. 8 represents a portion of the HTLV-III LTR containing regulatory regions.
The present invention is directed to the establishment of stable cell lines which express the tatIII gene. We have found that the prolific replication of the HTLV-III/LAV is in part caused by the presence of the tatIII gene. This gene regulates gene expression directed by the HTLV-III/LAV long terminal repeat (LTR) sequence (Sodroski et al., Science 227:171 (1985); Sodroski et al., ibid., 119,74 (1985); S. Arya et al., ibid., 69 (1985)). Because these tatIII cell lines do not contain the complete HTLV-III/LAV genome, it is possible to study the biochemical events associated with the tatIII gene without the possibility of virus spread.
We have discovered that cell lines which stably express the tatIII gene can be created by infection using a vector modified from the retroviral infector pZIPNEO (Cepko et al., Cell 37: 1053-1062 (1984).
The vectors used in the present invention can be in the form of plasmids or viral vectors such as those described in PCT/U.S. No. 85/00986 filed May 24, 1984. For example, the defective retroviral vector pZIPNEOSV(X)1 prepared as described by Cepko et al., supra contains Moloney murine leukemia virus LTR's, polyadenylation signals, sequences required for reverse transcription and for encapsidation of RNA, as well as the 5' and 3' splicing signals that normally produce subgenomic env gene messenger RNA. This vector also contains the bacterial gene for neomycin resistance (neo) which confers a dominant selectable resistance to the antibiotic G418 in eukaryotic cells (Southern, P. J. et al., J. Mol. Appl. Genet. 1: 327-341 (1982)) so that tatIII transfected cells can be identified. However, any indicator which can be identified can be used in a tatIII vector.
The HTLV-III/LAC tatIII gene was obtained from infectious proviral clone HXBC2 and encodes the HTLV-III/LAV associated trans-acting factor (See FIG. 1) (Arya et al., Science 229: 69-73 (1985); Sodroski et al., Science 229: 74-77 (1985)) This vector was prepared as described in FIG. 1.
DNA was introduced into the psi/2 (ecotropic) and psi AM (amphotropic) cell lines by the calcium phosphate coprecipitation method (Wigler et al., Cell 777-785 (1979). These lines constitutively produce the murine leukemia virus proteins but cannot package the viral transcripts (Cone, at el., PNAS 81: 6349-6353 (1984); Mann, et al., Cell 33: 153-159 (1983)). Two days following transfection, cells were selected with the antibiotic G418 (400 μg/ml for fibroblast lines and 700 μg/ml for lymphocytes). G418 resistant clones were evident in 7 to 10 days. Insertion of the tat regions does not interfere with splicing events required for transcription of the neo genes. G418-resistant psi 2 and psi AM clones were isolated and the virus from clones producing greater than 103 infectious units per ml were used to infect the test cells. (King et al., Science 228: 554-558 (1985)). Cells resistance to G418 were observed subsequent to infection of all the cell lines tested.
We have found that by substituting the Moloney LTR with other modified LTR's a tissue specific expression vector can be obtained. The vectors are constructed using a tissue specific enhancer(s) operatively positioned in the same sequence with a heterologous DNA segment corresponding to the polypeptide of interest, as well as a stop codon and polyadenylation sequence downstream (3') from that gene. The vector should also contain a replication origin.
The vector contains at least the segment of an enhancer which determines the tissue specificity of that enhancer, hereinafter referred to as the "tissue specificity determinant." The vector preferably contains a complete viral enhancer, rather than just the tissue specific determinant from such an enhancer and preferably the tissue specific determinant is part of the complete enhancers.
The promoter contained in the vector can be any of the known promoters which function to permit expression of a desired product in the host of choice. Preferably the promoter is a viral promoter from the same class of virus as the enhancer. The preferred class of virus is retrovirus, and the preferred viruses for use in conjunction with the invention are the Akv, SL3-3, and Friend viruses.
The term "tissue specific" as used in this disclosure and claims, means that the vector operates to produce a greater amount of desired product in the targeted tissue than it does in other tissues under normal culture conditions. Tissue specific vectors may produce 1.5 to 1,000 or more times as much expression product in the target tissue as in other tissues. These tissue specific expression vectors are more fully described in PCT/U.S. No. 85/00986 which is incorporated by reference.
The tissue specific determinant can be homologous, meaning it came from the same virus as the promoter, or heterologous, in which case it is not from the same virus as the promoter. Heterologous tissue specific determinants can be excised from other viral systems, or can be synthesized using known techniques. Tissue specific determinants which are specific to the target tissue can be identified by assay techniques, where vectors encoding an indicator or marker compound, e.g., chloramphenicol acetyl transferase (CAT), an indicator which can be easily quantified as described below, to determine which vectors are effective in the tissue.
If desired, enhancer(s) from tissue specific vectors can be compared in DNA sequence to the enhancers which are not specific to the target tissue to determine the DNA sequence of the tissue specific determinant. Thereafter, at least the tissue specific determinant, preferably the entire enhancer, may be utilized in the desired vector containing the tatIII gene and the resulting tissue specific vectors utilized to express this gene product in the tissue of choice.
Various cell lines differ in their ability take up and express the transfected "tatIII " DNA. We have found that Raji cells, HUT 78 cells, Jurkat cells, HeLa cells and NIH 3T3 cells are useful. Human T-cells and B-cells, generally are very useful. Another useful method of achieving transfection with the "tatIII " DNA is to use cells infected with either HTLV-I or HTLV-II.
To test for the presence of tatIII gene transcripts, the total cellular RNA was extracted from several clonal isolates of infected NIH 3T3 and HeLa cells. FIG. 2A demonstrates that tatIII gene transcripts were present in the respective infected cells and absent from uninfected controls. This demonstrates that it is possible for the tatIII gene and the neo gene to be transcribed by use of these vectors.
To determine whether the tatIII gene is functional, the tatIII cell lines of the present invention were transfected with plasmid pU3R-III (FIG. 3). This plasmid contains HTLV-III/LAV LTR sequences 5' to the CAT gene (See U.S. patent application Ser. No. 614,297). In PCT/U.S. No. 85/00985 filed May 24, 1985, we demonstrated that the tatIII gene product activates in trans gene expression directed by the HTLV-III/LAV LTR.
The level of HTLV-III/LAV LTR directed gene expression was dramatically increased in ZIPNeotatIlI-infected HeLa cells compared with uninfected cells as shown in FIG. 2B. See also Table 1. For example relative to pSV2CAT, gene expression directed by plasmid pU3R-III, was increased between 760 and 1100 times depending upon the clone tested. In ZIPNeotatIII-infected Raji cells, HTLV-III/LAV LTR-directed gene expression was increased approximately 2000-fold relative to the control of uninfected Raji cells. The level of CAT activity was increased by 6.5 to 23 times in infected NIH 3T3 cells. The levels of Rous sarcoma virus LTR-and HTLV-I LTR-directed CAT gene expression were the same in infected and in uninfected cells.
TABLE 1__________________________________________________________________________Effect of ZIPNeotatIII infection on HTLV LTR-directed gene expression. Plasmid Relative CAT Activitya PU3R-I PU3R-II PU3R-IIIVirus Cell Lineb pSV2CAT Stimulationc Stimulationc Stimulationc RSVCAT__________________________________________________________________________None Raji 1.00 0.62 0.24 0.8ZIPNeotatIII Raji-P 1.00 0.68 530.00 (2208) 1.40ZIPNeotatIII Raji-C1 1.00 650.00 (2708)ZIPNeotatIII Raji-C2 1.00 427.00 (1779)None Hela 1.00 0.50 0.12 6.00ZIPNeotatIII Hela-P 1.00 0.61 115.00 (958) 3.00ZIPNeotatIII Hela-C1 1.00 135.00 (1125) 8.50ZIPNeotatIII Hela-C1 1.00 92.00 (766) 5.70ZIPNeotatIII NIH3T3-P 1.00 4.70 (23) 5.20ZIPNeotatIII NIH3T3-C1 1.00 1.30 (6.5) 4.20ZIPNeotatIII NIH3T3-C2 1.00 2.20 (11) 6.20__________________________________________________________________________
The presence of functional tatIII proteins was determined by measuring the level of HTLV-I(pU3R-Ind HTLV-III/LAV (pU3R-III) LTR-directed CAT gene expression following transfection of the respective infected cells. The levels of CAT activity obtained following transfection of uninfected (-) and infected (+) cells with the control plasmid pSV2CAT are also shown. The autoradiograms of FIG. 2B show the level of enyzme activity present in a typical reaction for the time (numbers underneath CAT assays) indicated. Unreacted chloramphenicol (Cm) and acetylated reaction products (AcCm) are also shown.
These experiments demonstrate that we have established cell lines which will stably express the tatIII gene product. Moreover, the level of trans-activation in many of these infected cells approximates the level of transactivation obtained with infection by the actual HTLV-III/LAV virus.
The use of these cell lines, which express high levels of tatIII genes, permits the development of high-level gene expression systems. For example, the desired genes under control of the HTLV LTR can be introduced either transiently or stably into the tatIII cell lines, which should result in the expression of a large quantity of the desired gene product. Such a system, would be useful for the overproduction of the HTLV-env proteins, which in turn, could be used clinically for diagnostic or prophylactic purposes.
The use of the tatIII cell lines of the present invention also permit the large scale production of the tatIII protein.
Using these tatIII cell lines permits the large scale production of other tatIII gene products, as well as the tatIII protein.
We have now discovered the tatIII protein which is approximately 14,000 daltons molecular weight comprising 86 amino acids and exhibits trans-activating activity. Although the region of the HTLV-III/LAV genome necessary for trans-activation of the HTLV-III/LAV LTR had been determined and shown to contain an open reading frame capable of encoding a protein of 86 amino acids in two exons (FIGS. 4 and 7), the trans-activator protein itself had not been identified until now. The protein we have discovered migrates with an apparent molecular weight of 14kD, is syntehesized from the HTLV-III/LAV transactivator gene and is immunoreactive in some individuals infected with HTLV-III/LAV.
The first coding exon of the HTLV-III/LAV transactivator gene is shown in FIG. 7. It lies 5' to the envelope gene (from nucleotide 5365 to 5607). A single ATG codon at position 5411 could potentially initiate the transactivator protein, tatIII. The second coding exon is present in an alternate reading frames within the env gene. Doubly-spliced RNA molecules containing these exons have been detected in virus producing cell lines J. Sodroski et. al., Science 227:171 (1985); J. Sodroski et. al., ibid.: 119,74 (1985); S. Arya et. al., ibid.: 69 (1985)!. M. Popovic et. al., Science 224:497 (1984)!.
To establish that this open reading frame encodes a functional protein product that is antigenic in patients, 221 nucleotides of the predicted first coding exon from nucleotide 5391 to 5611 were inserted into an open reading frame vector (ORF) containing the bacterial beta-galactosidase gene G. Weinstock et. al., PNAS 80,4432 (1983)!. Both promoter and translation initiation signals are provided by the ompF gene, an E. coli gene encoding an abundant outer membrane protein. A plasmid containing the tatIII region was constructed so as to create a continuous open reading frame between the ompF and beta-galactosidase genes (ompf-and tatIII -beta-galactosidase, pBGT-III) (FIG. 5). A plasmid containing an in-frame fusion product between the ompF-beta-galactosidase, genes without an insert was also constructed (ompF-beta-galactosidase, pBG).
Cells containing the pBGT-III plasmid express an approximately 120 kd fusion protein that can be detected by immunoprecipitation with rabbit anti-beta-galactosidase serum (FIG. 6A). The rabbit anti-beta-galactosidase serum also immunoprecipitates a smaller protein of the expected 116 kd sizes from cells transformed by the pBG plasmid alone. To determine if the fusion protein is immunogenic in patients, a panel of serum from people seropositive for HTLV-III/LAV was used to immunoprecipitate extracts derived from bacteria containing the plasmids pBG or pBGT-III. Of twenty tested serum samples (five of which are shown in FIG. 6A), one was positive (38-1). This serum specifically immunoprecipitated the pBGT-III fusion protein but did not react with the product of the pBG plasmid, indicating that the antigenic determinants recognized by the serum were probably provided by the HTLV-III/LAV insert. This shows that in at least some patients infected with HTLV-III/LAV, the tat-III gene can encode an immunogenic product.
To determine if the positive antiserum, 38-1, could identify a protein encoded by the tatIII gene, 35 S!cysteine labelled whole cell lysates of H9 cells infected with HTLV-III/LAV (3) Popovic et. al., supra! were used in immunoprecipitation analysis. As a control, a patient serum that was non-reactive with the pBGT-III fusion protein, serum 4-3, was also used. FIG. 6B shows the immunoprecipitation of HTLV-III/LAV infected cells. H9/HTLV-III cells Popovic et. al, supra! were labelled overnight with 100 uCi per ml of 35 S! cysteine and reacted with a patient antiserum (38-1) that recognizes the ompF-tatIII -betagal product (lane 1) or a patient antiserum that failed to recognize the ompF-tatIII -betagal product (4-3). 14 C! labelled protein markers (Amersham) are shown in, the leftmost two lanes, (k=kDa; p, protein; gp, glycoprotein). Both the 38-1 and 4-3 sera recognize the viral envelope glycoproteins gp160/120, the viral gag gene precursor, p55 and smaller viral proteins, including the structural core proteins p24 and p17. However, a 14 kD protein recognized by the 38-1 antiserum is not present in the immunoprecipitates with the 4-3 antiserum and has no reaction. Neither serum immunoprecipitated this protein from uninfected R9 cells. These observations confirm that the 14 kD protein is the product of the bacterial fusion tatIII protein.
As further proof that the 14 kD protein is indeed encoded by the trans-activator gene of HTLV-III/LAV, it should be present in cells that express only the tatIII gene. Thus, HeLa cells were transfected with a deletion mutant of the HTLV-III genome that had previously been designated p (83-5365/8053-9296) () Sodroski et. al, supra! (FIG. 4). Cell lines were established by co-transfection with a plasmid that encodes a gene for resistance to the antibiotic G418. This cell line stably expresses the HTLV-III/LAV trans-activator gene as judged by an enhanced rate of HTLV-III/LAV LTR directed expression of the chloramphenicol acetytransferase (CAT) gene (Table 2). This observation is consistent with previous experiments that show that this plasmid encodes the trans-activating factor.
Immunoprecipitation of the transfected cell line with the 38-1 sera shows a 14 kD protein that is absent in the untransfected Hela cells as shown in FIG. 6C. Clones were selected for resistance to G418. Cells were then labelled overnight with 100 uCi of 35 S!cysteine per ml and total cellular lysates used for immunoprecipitation with either patient anti-serum 38-1 (lane 1) or 4-3 (lane 2). Lanes 3 and 4 are immunoprecipitates of untransfected Hela cells with 38-1 and 4-3 respectively (k=kDa).
The serum 4-3 that does not immunoprecipitate the tatIII -beta-galactosidase fusion protein also failed to recognize the 14 kD protein. As the plasmid used for transfection had been deleted for all of the gag, pol, sor and 3' ORF coding regions of the HTLV-III/LAV genome, the 14 kD protein cannot be encoded by these regions.
Lymphocytic cell lines were also established by infecting HUT78, a human T-cell line and Raji, a human B-cell line with a retroviral vector, pZIPNeo tatIII that was constructed to allow expression of the tatIII gene (FIG. 4). The retroviral vector does not contain the gag, pol, sor or 3' ORF open reading frames. In each case, a 14 kD protein that is absent in the respective uninfected parent cell lines is immunoprecipitated with serum 38-1 but not with serum 4-3 (See FIG. 6D). Patient serum 38-1 was used to react with lysates from Raji ZIP tatIII (lane 1), uninfected Raji cells (lane 2), 2 different clones of HUT78 ZIP tatIII (lanes 3 and 4) and uninfected HUT78 (lane 5) (k=kDa).
Since a truncated envelope gene is present in the plasmids p Δ(83-5365/8053-9296) and pZIPNeo tatIII, deletion mutants that had previously been shown to trans-activate the HTLV-III/LAV LTR but do not contain a substantial portion of the env gene were used in transfection assays. Raji cells were transiently transfected with plasmids p Δ(83-5365/8053-9296) and p Δ(83-5365/6617-7198/8053-9296) which contains a major internal deletion within the env gene (FIG. 4). Inmmunoprecipitation of cells transiently transfected with both plasmids with serum 38-1 similarly shows a 14 kD protein that is absent in untransfected Raji cells.
Although it is believed that the tat-III gene is encoded by two exons, deletion analysis demonstrated that only the first coding exon is required for tatIII activity Sodroski, supra!. Specifically, a plasmid, p Δ(83-5365/5607-7719/8053-9296), that contains only the first 69 amino acids of the first coding exon and deletes most of the envelope gene was shown to retain tatIII activity. (See FIG. 6E). Raji cells were transfected with p Δ(83-5365/8053-9296), p Δ(83-5365/6617-7198/8053-9296) or p Δ(83-5365/5607-7719/8053-9296) using DEAE-dextran as described 4. Sodroski, supra 8. Queen et al., Cell 33:729 (1983!. Cells were harvested after 48 hours and labelled with 100 uCi per ml of 35 S!cysteine overnight. Total cellular lysates were then reacted with patient antiserum 38-1. Cold Raji lysates were used as competitor to reduce background during immunoprecipitation. Lanes 1-4 show immunoprecipitation of untransfected Raji cells, cells transfected with p Δ(83-5365/8053-929), p Δ(83-5365/6617-7198/8053-9296) and p Δ(83-5365/5607-7719/8053-9296) respectively (See FIG. 6E). Immunoprecipitation of cells transiently transfected with this plasmid reveals a smaller protein of approximately 12 kD, a shift in size consistent with the expected loss of 17 amino acids.
This 14 kD protein is present in all cells tested that demonstrate trans-activation of HTLV-III/LAV LTR directed gene expression. The protein is also expressed in cell lines that contain plasmids constructed so as to express specifically proteins encoded by the tatIII gene. These cells contain portions of the HTLV-III/LAV genome in which the entire gag, pol, sor and 3'ORF genes have been deleted. Finally, the 14 kD protein is present in cells containing the HTLV-III/LAV genome that carries substantial deletions in the env gene as well. Truncation of the tatIII gene results in a more rapidly migrating protein. Although the immunoprecipitated protein is somewhat larger than its predicted size (10 kD), this may be due to post-translational modification, or alternatively, to anomalous migration associated with highly basic and proline-rich polypeptides.
The use of this protein can dramatically increase the rate of transcription of a heterologous gene product and hence the expression of the desired gene product.
The present invention is further illustrated by the following examples. These examples are provided to aid in understanding of the invetion and are not to be construed as a limitation thereof.
Construction of Vector Used to Establish TatIII Cell Lines
The defective retroviral vector pZIPNEOSV(X) developed by Mulligan and coworkers (Cepko, et al., supra 1984) was used to construct a vector for establishing stable tatIII cell lines. This vector contains Moloney murine leukemia virus LTRs, polyadenylation signals, sequences required for reverse transcription and for encapsidation of RNA as well as the 3' and 5' splicing signals that normally produce subgenomic RNA. In addition, the vector contains the bacterial neomycin (neo) reistance gene that confers a dominant selectable resistance to the antibiotic G418 in eukaryotic cells (Southern and Berg, supra 1982). The tat gene of HTLV-III was isolated from the plasmid shown in FIG. 1 by cleaving the DNA at the indicated restriction sites. The HTLV-III/LAC tatIII gene was obtained from infectious proviral clone HXBC2 and encodes the HTLV-III/LAV associated trans-acting factor (See FIG. 1) (Arya et al., Science 229: 69-73 (1985); Sodroski et al., Science 229: 74-77 (1985)).
Transfection of Cell Line with TatIII Vector
DNA was introduced into the psi/2 (ecotropic) and psi AM (amphotropic) cell lines by the calcium phosphate coprecipitation method (Wigler et al., Cell 16: 777-785 (1979). These lines constitutively produce the murine leukemia virus proteins but cannot package the viral transcripts (Cone, at el., PNAS 81: 6349-6353 (1984); Mann, et al., Cell 33: 153-159 (1983)) . Two days following transfection, cells were selected with the antibiotic G418 (400 μg/ml for fibroblast lines and 700 μg/ml for lymphocytes). G418 resistant clones were evident in 7 to 10 days. Insertion of the tatIII regions did not interfere with splicing events required for transcription of the neo genes. G418-resistant psi 2 and psi AM clones were isolated and the virus from clones producing greater than 103 infectious units per ml were used to infect the test cells. (King et al., Science 228: 554-558 (1985)). Cells resistance to G418 were observed subsequent to infection of all the cell lines tested.
Construction of TatIII Fusion Product
Construction of a tatIII fusion product in a bacterial open reading frame vector is shown in FIG. 5. About 221 nucleotides of the first coding exon of tatIII (nucleotide 5391 to 5611) were inserted into the Sma I site of pORF1 (6) Ratner et. al., Nature 313:227 (1985)!. Shown below are the frames of the ompF gene, the tatIII gene and the lacz gene. Plasmids were initially selected in E. coli strain MH3000 by scoring for blue lacZ+ colonies on LB-ampicillin plates containing 5-bromo-4-chloro-3-indoxyl-beta-D-gal Ratner et. al, supra!. Plasmids were then transferred to E. coli TK1046, a strain that permits higher expression of the ompF promoter.
Expression of TatIII Product
Expression of the fusion ompF-tatIII -betagalactosidase protein is shown in FIG. 6A. Tk1046 cells containing pBGT-III or pBG were grown in L broth at room temperature until the optical density at 550 nm was approximately 0.3 to 0.4. They were then labelled in M63 glucose medium with 100 uCi of 35 S!cysteine per ml for 15 minutes. Labeled proteins were processed for immunoprecipitation as previously described (6) G. Weinstock, et. al., PNAS 80:4432 (1983)!. Lanes 1-6 are immunoprecipitates of the ompF-betagalactosidase product from pBG with rabbit anti-beta-galactosidase serum, patient anti-serum 4-3, 11-2, 12-4, 13-4 and 38-1 respectively. Lanes 7-12 are immunoprecipitates of the ompF-tatIII -betagalactosidase protein from pBGT-III with rabbit anti-beta-galactosidase serum, patient serum 4-3, 11-2, 12-4, 13-4 and 38-1 respectively (k=kDa)
Deletion analysis
(a) Structure of HTLV-III/LAV deletion mutants that retain trans-activating ability is shown in FIG. 5. The restriction map of the complete HTLV-III/LAV provirus in plasmid pHXBc2 is shown on the topmost line. The second line drawing depicts the splicing pattern of the message coding for the potential, tatIII gene product based on the sequence of CDNA messages from HTLV-III/LAV infected cells ( Sodroski et al., Science 227, supra; Sodroski et al., ibid 119, supra; Arya et al., supra; Mussing et al., Nature 313:450 ( 1985!) Construction of the deletion mutants is described below. Numbers corresponding to the deletion endpoints are based on the sequence of Ratner et al. supra where the RNA cap site is designated as +1. Zigzag lines, mark the polyadenylation and splice signals from the SV40 small t-antigen coding region (H=Hind III; S=Sal; Bg=Bgl/II; Bm=Bam HI).
(b) To construct the deletion DNAs plasmid pU3R-I7 was first cleaved at the unique site Sma I site (position -322) within the LTR. Digested DNA was incubated with the exonuclease Bal 31. At various time points aliquots were removed and the DNA was phenol chloroform extracted then ethanol precipitated. Any protruding ends were filled with T4 polymerase followed by ligation to Xho I linkers. After linker addition the DNA was recircularized and transfected into E. coli. strain HB101. Several size selected clones were characterized by restriction enzyme analysis. The desired clones were then end labeled at the Xho I site and subjected to Maxam and Gilbert sequence analysis (see Maxam, A. M. et al., Proc. Natl. Acad. Sci, USA, 74: 560-564 (1977)) to determine the end points. The name of each plasmid is indicative of the number of nucleotides present relative to the transcription start site.
(c) The deletion DNAs were transfected into the following cell types by a modification of the DEAE dextran coprecipitation; technique (see Queen C. et al., Cell, 33: 729 (1983)). Hela, a human epithelial cell line; HUT 78, an OKT4+ helper/inducer human T cell line derived from an HTLV negative patient with sezary syndrome (see Gey, et al., Cancer Res., 12: 264 (1952)); NC37, a B lymphocyte line established from a normal donor and immortalized by Epstein Barr virus (see Manzari, V. et al., Proc. Natl. Acad. Sci. USA, 80: 11-15 (1983)); and C81-66/45, derived by fusion of primary umbilical cord blood cells with an HTLV-I producing cell line (see Salahuddin, S. Z. et al., Virology, 129: 51-64 (1983)). This cell line does not produce virus but contains the HTLV-I associated trans-acting factor. Forty eight hours following transfection cellular extracts were made and CAT assays were performed as described. The values shown are normalized to the CAT activity present in similar cells transfected with a control plasmid pSV2CAT taken as 1.0. The bracketed number indicates the relative CAT activity in infected cells compared with uninfected cells normalized to the activity present in the uninfected lymphocyte lines transfected with the pSV2CAT plasmid that contains the SV40 enhancer promoter sequences (Laimins et al., supra).
TABLE 2______________________________________Effect of HTLV III/LAV tat gene expression on HTLV III-LAV LTRdirected gene expression.PlasmidsbRelative CAT ActivitycCell Linesa pSV2CAT pU3R-III______________________________________Hela 1.0 .10Hela III extat 1.0 67 (670)Raji 1.0 .24Raji ZIONEOtatIII 1.0 650 (2708)HUT 78 1.0 .60HUT/78ZIPNEO/tatIII 1.0 330 (550)______________________________________ a. The Raji ZIPNEO tatIII and HUT78 ZIPNEO tatIII cell lines were established by infection with the retroviral tatIII expression vector pZIPNEO tatIII as previously described (10). the Hela IIIextat line was established via cotransfection with plasmids IIIextat (formerly pΔ835365/8053-9296) and pU3RIIINEO) b. Plasmid pSV2CAT contain the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the SV40 early region promoter sequences C. Gorman, Moffat, L. F. and Howard, B. Mol. Cell. Biol. 2, 1044 (1982). Plasmid pU3RIII contain HTLVIII/LAV LTR sequences directing expression of the CAT gene. c. Cells were transfected with 2 ng of plasmid DNA and CAT assays were performed 48 hours posttransfection. The CAT activity is normalized to the activity obtained with pSV2CAT in the same cell. The number in parenthesis represents the fold stimulation in the infected as compared t the uninfected cells.
The present invention permits high levels of production of proteins and like-materials expressable by genes.
There are genetic segments of the HTLV-III LTR region which in the presence of the tatIII gene product, result in high rates of gene expression. These segments are called TAR elements and can be utilized to provide a trans-activated response. The location of this TAR element has been mapped to lie between nucleotides -17 and +80 of the HTLV-III genome as shown in FIG. 8 Rosen, C. A. et al., Cell 41: 813-823 (1985)!. However, when only this portion of nucleotide segment is used, it must be provided with another enhancer upstream to provide trans-activation for the tatIII product. More preferably, the nucleotide region ranging from -167 through +80 is used. This nucleotide region contains an enhancer sequence, between -137 and -17.
By using a vector containing a TAR sequence, and a desired heterologous gene and transfecting the tatIII cell lines of the present invention, it will be possible to result in trans-activating the tatIII cell line, thereby producing high yields of the desired heterologous gene product. Alternatively, use of the the tatIII protein of the present invention can be trigger trans-activation, thereby achieving high levels of expression of the desired gene product. This can be accomplished by adding the tatIII protein to a culture medium, wherein a cell has been transfected with a predetermined heterologous gene and a TAR element.
One can use a vector containing only the TAR element and a tissue specific enhancer to result in transactivating only specific cells. For example, one could use the enhancer for the immunoglobulin gene to result in activating only B lymphocyte cells.
The present invention also permits the use of an improved process for detecting the presence of HTLV-III/LAV virus in individuals. Current assay techniques require culturing lymphocytes from individuals. This is a time consuming and costly process. In addition, the sensitivity of current assay techniques will not always detect the presence of the virus.
We have discovered that by using the tatIII cell lines of the present invention, a sensitive and efficient assay method is possible. Preferably, one takes lymphocytes or whole blood and adds it to a culture medium containing the tatIII cell lines of the present invention, but other "fluid" can be used. More preferably one uses lymphocytes. These cells are then incubated at 37� C. and watched to determine whether or not a cytopathic effect is exhibited in the tatIII cell line.
Preferably, one uses a tatIII cell line established from a cell line that is particularly sensitive to the cytological property of the HTLV-III/LAV virus. Such cell lines include HUT 78 cells, C8161 cells and Jurkat cells.
Preferably, the cell lines contain a marker that is released upon the cells death. This marker can be used to determine the cytopathic effect of the tested material. Thus, when a cell dies, the marker is released into the culture medium resulting in a reaction with the medium that is visually observed. Examples of such markers include chromium.
This assay system allows for the detection of the HTLV-III/LAV virus in periods of time typically as short as three days as opposed to the normal two weeks that it takes with using reverse-transcriptase. Cultivation and incubation techniques used are those well known to anybody of ordinary skill in the art.
The tatIII cell lines of the present invention also permit the development of a system for screening for a compound that will mitigate the cytopathic effects of the HTLV-III/LAV virus. Using these tatIII cell lines, two types of transfected cell lines are created to detect the differential response of the cells. In both types, the cell is transfected with a vector containing a heterologous gene, whose gene product is selectable for example, the CAT gene. In the first situation, the expression of the heterologous gene product is under the control of an HTLV-III TAR element, more preferably the full HTLV-III LTR, and thus the level of its expression is effected by the functioning of the tatIII gene product. In the second cell line, the heterologous gene is under the control of a separate regulatory sequence, and the level of expression of the selectable gene product is independent of tatIII function. Thereafter, a compound to be screened is added to each cell type by standard techniques in increasing concentrations. The expression of the selectable gene is measured by techniques well known in the art to determine which compounds inhibit tatIII function without being toxic to the cell itself.
It is preferable to screen compounds that prevent the interaction of the tatIII protein with the sequences responsive to the tatIII protein in the HTLV-III LTR or prevent the ability of the tatIII protein to trans-activate the HTLV-III LTR.
We have found that in cell lines, exhibiting trans-activation by the tatIII protein and thus, high levels of protein expression, there is a negligible increase in the levels of corresponding messenger RNA. Consequently, using compounds that inhibit translation, such as substances that affect the formation of translational initiation complexes or alter the bonding of ribosomes to the viral mRNA is most preferable.
Examples of compounds that can be used in this screening process include competitors, compounds that inhibit translation and compounds that alter the binding ability of a compound. Compounds such as those described in the Physicians' Desk Reference, 38th ed. Medical Economics Co., Droden, N.J. (1984), that can be used in the present screening process can be readily determined by the person of ordinary skill in the art based upon the above disclosure.
A preferred group of competitors would be mutant tatIII proteins that would retain their ability to bind to nucleic acid but are deficient in some other trans-activation function. Such proteins should serve as efficient competitors for functional tatIII proteins. Random mutagenesis by, for example, chemical modification can be used to generate large numbers of tatIII mutants without a specified target region. In one embodiment, one would use the first coding exon of tatIII which can be isolated using convenient restriction endonuclease sites. This region will be cloned into the replicative form of phage M13.
Single stranded M13 containing the tatIII insert in either orientation is mutagenized using methoxyalamine. This can generate single and double nucleotide substitutions at a frequency of greater than 50% (Kadonaga and Knowles, Nucl. Acids Res, 13: 1733 (1985). The single stranded DNA of a clone in one orientation is annealed to that of a clone in another orientation so that a double stranded insert is reconstituted. The chemically modified inserts are removed from vector M13 DNA by restriction digestion and recloned into cut alkaline-phosphatase treated M13 replicative form DNA. Clones containing inserts are identified by the colorless plaques generated when the insert disrupts the beta-galactosidase gene present in the M13 vector. The insert fragment can then be sequenced using the dideoxy method of Sanger et al., PNAS, 74: 5463 (1977).
Following generation and sequencing of tatIII mutants in M13 by the methods described above, the insert fragmets are recloned into an HTLV-III expression vector containing HTLV-III LTR and a selectable heterologous gene, such as CAT and transfected into eukaryotic cells. The activity of the mutant tatIII proteins will be determined by testing their ability to trans-activate the HTLV-III LTR in a co-transfection assays. Using the transient CAT assays as many as 100 plasmid clones can be tested for activity in the period of one week. Moreover, mutations that increase or decrease the trans-activating ability can be detected in a quantitative manner.
Those mutants that are no longer able to trans-activate will be tested in the above-described screening process. If a mutant tatIII protein that can effectively compete with the active form is found then the mutant tatIII gene will be subcloned into the retroviral vector.
The tatIII protein of the present invention can also be used to develop anti-sera which could effectively block infection by HTLV-III/LAV virus. We have found that a fusion protein containing this protein is immunoreactive with patient serum (See FIG. 6). The protein is used as an immunogen for raising polyclonal and monospecific antibodies tatIII protein of the present invention can be used to immunize rabbits or goats either as pure protein or a betagalactosidase fusion product (See FIGS. 5 and 6).
The effectiveness of the anti-sera can be tested by techniques well known to the person of ordinary skill in the art. For example the tatIII anti-sera (or purified IgG fraction) together with the appropriate plasmids is injected into Xenopus oocytes. The HTLV-III LTR transcriptional regulatory sequences direct high levels of CAT gene expression in this system and because oocytes synthesize large quantities of RNA and protein following microinjection this system is better suited than microinjection into cells.
The tatIII anti-sera is introduced into the oocytes together with the pU3R-III plasmid (HTLV-III LTR upstream of CAT) and several different tatIII gene expresser plasmids. Control experiments using plasmids unresponsive to tatIII and normal serum will be done in parallel in an analogous manner to the screening process described above. A decreased level of CAT gene expression directed by plasmid pU3R-III and the tatIII expresser plasmid in the presence of the tatIII specific anti-sera indicates inhibition of trans-activation.
It is evident that those skilled in the art, given the benefit of the foregoing disclosure, may make numerous other uses and modifications thereof, and departures from the specific embodiments described herein without departing from the inventive concepts, and the present invention is to be limited solely by the scope and spirit of the appended claims.
|Arya, et al., Science 229:69 73 (1985).
|Arya, et al., Science 229:69-73 (1985).
|Cepko, et al., Cell 37:1053 1062 (1984).
|Cepko, et al., Cell 37:1053-1062 (1984).
|Cone, et al., PNAS 81:6349 6353 (1984).
|Cone, et al., PNAS 81:6349-6353 (1984).
|D. Klatzmann, et al., Nature (London) 312:767 (1984).
|D. Klatzmann, et al., Science 225:54 (1984).
|F. Barre Sinoussi, et al., Science 200:868 (1983).
|F. Barre-Sinoussi, et al., Science 200:868 (1983).
|F. Siegal, et al., ibid. 1439.
|G. Shaw, et al., Science 227:177 (1985).
|G. Weinstock, et al., PNAS 80:4432 (1983).
|Gaynor, et al., Proc. Natl. Acad. Sci. USA, 81:1193 1197 (1984).
|Gaynor, et al., Proc. Natl. Acad. Sci. USA, 81:1193-1197 (1984).
|H. Lane, et al., ibid., 309:453 (1983).
|H. Masur, et al., ibid. 1431.
|Hahn, et al., Nature 312:166 168 (1984) (cited by Examiner).
|Hahn, et al., Nature 312:166-168 (1984) (cited by Examiner).
|J. Schupback, et al. ibid. 503.
|J. Ziegler, et al., ibid. 311:565 (1984).
|King, et al., Science 228:554 558 (1985).
|King, et al., Science 228:554-558 (1985).
|M. Gottlieb, et al., N.E. J. Med. 305:1425 (1981).
|M. Popovic, et al., Science 224:497 (1984).
|M. Seligman, et al., N.E. J. Med. 311:1286 (1984).
|Mann, et al., Cell 33:153 159 (1983).
|Mann, et al., Cell 33:153-159 (1983).
|Marx, et al., Science 224:475 477 (1984) (cited by Examiner).
|Marx, et al., Science 224:475-477 (1984) (cited by Examiner).
|R.C. Gallo, et al., ibid. 224:500 (1984).
|Sarngadharan, et al., ibid. 506.
|Sodroski, et al., (b) Science 225:381 (cited by Examiner).
|Sodroski, et al., Science 119:74 (1985).
|Sodroski, et al., Science 227:171 (cited by Examiner).
|Sodroski, et al., Science 229:74 77 (1985).
|Sodroski, et al., Science 229:74-77 (1985).
|Southern, P.J., et al., J. Mol. Appl. Genet. 1:327 341 (1982).
|Southern, P.J., et al., J. Mol. Appl. Genet. 1:327-341 (1982).
|Wigler, et al., Cell 16:777 785 (1979).
|Wigler, et al., Cell 16:777-785 (1979).
|Citing Patent
|Filing date
|Publication date
|Jun 23, 1999
|Sep 28, 2004
|Uab Research Foundation
|Cell-based assay for immunodeficiency virus infectivity and sensitivity
|Apr 28, 2010
|Jan 24, 2012
|Iris Engines, Inc.
|Radial impulse engine, pump, and compressor systems, and associated methods of operation
|Sep 8, 2004
|Feb 10, 2005
|Uab Research Foundation
|Cell-based method and assay for measuring the infectivity and drug sensitivity of immunodeficiency virus
|U.S. Classification
|435/5, 435/6.13
|International Classification
|Cooperative Classification
|European Classification
|Dec 20, 2001
|Fee payment
Year of fee payment: 4
|Feb 21, 2006
|Fee payment
Year of fee payment: 8
|Mar 1, 2010
|Fee payment
Year of fee payment: 12


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De Pere Jobs
Looking for jobs in De Pere? Snagajob has great opportunities with the area's leading companies. And applying for De Pere jobs has never been easier. Look at the job listings below, click on a job title that interests you and hit the "apply now" button. Part-time jobs, teen jobs, student jobs and summer jobs are now just a click away. Start your job search now.
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Hungry for Insight to Continually Improve Service
Starting each day by reading customer feedback allows Chuck Sliker to get extensive insight into customers' likes and dislikes. Sliker, senior vice president of operations services at Arby's, uses that information to strengthen weak points in the company's products or service, as well as to commend individual restaurants and specific associates for superior customer service.
With a firm belief that the customer's own words afford companies significant opportunities to improve, Sliker introduced Arby's
Promise Check in 2008, adding to the numerous programs-including inspection reports-the company uses to gather feedback about its nearly 3,600 restaurants. Sliker and his team worked with Mindshare Technologies to customize an already existing program into one that met Arby's unique needs. The new approach focuses on gathering real-time, actionable customer feedback.
Since its launch, the program has allowed the fast-food chain to amass more than 10 million responses. "We know every single morning, and sometimes within hours, about what happened, where it happened, and who was involved," he says, adding that the extensive data from Arby's Promise Check gives the organization deep insight into customers' perceptions on everything from speed of service to the level of hospitality they receive when choosing Arby's.
"There is no more motivating, actionable data point than the customer's own words," Sliker says. "We can all say that we think we are the best operators and know our businesses extremely well, but the bottom line is that customers tell you every single day what opportunities there are for you to improve your business. The day you stop listening to your customers is the day you start on a very slippery slope."
Customer feedback is regularly shared with frontline employees as an inspiration to give exceptional service. Sliker says that by showcasing "the many wonderful stories of great service," Arby's helps provide examples of behavior that associates should emulate.
Negative feedback is also shared with employees, including Arby's training department, which uses it in the development of its programs.
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Packaging Engineer - RLC 2385
RLC Recruitment Co., Ltd.
- Double check packaging spec and graphic compare with current packaging and product to reduce complexity and avoid any mistake.
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- Bachelor Degree of Packing Engineering or equivalent
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- Experience 3-5 year
- Male/Female 28-32 years old.
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Please send your latest update resume in word document with a recent photo to us and you'll be contacted for full detail of the job and the interview if your profile is qualified for the job. Please quote position title AND reference code RLC- 2385 in email subject line, Thank you.
RLC Recruitment Co., Ltd
142 Two Pacific Place, 18th Floor, Sukhumvit Road, Bangkok 10110 Thailand
For more information please visit our website: www.rlc-asia.com
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RLC was recently established to address the needs of the growing number of companies looking for a customized and personal approach to recruitment.
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Liliana is the Lead Instructor in Second and Foreign Languages at the Larimer Campus in Fort Collins. She coordinates and teaches in the Foreign Languages Program, the English as a Second Language Program, and the Teaching English as a Second Language (TEL) Certificate Program (Larimer Campus). She is a native Spanish speaker from Mendoza, Argentina. Her academic area of interest is second and foreign language methodology, and, more recently, educational policy issues. Liliana is a Fulbright Scholar and holds a doctorate degree in Educational Leadership.
¡Hola! I am interested in the theory and practice of best teaching methods and I pursue research in this area with the hope that more efficient teaching delivery would serve our students better. I believe that learning another language and getting acquainted with other cultural beliefs and traditions is one of the most important tools to get by in today's global society. While most of my teaching experience has been in the traditional classroom, I strongly believe in providing students with alternative learning opportunities. Online classes fulfill this purpose. Currently, there is abundant technical assistance to make online teaching and learning a successful endeavor.
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Top Definition
A chinese restaurant located in SF.
Dude: Hey! Fuk U?
Badass goodlooking piece of a hot woman: Fuck off! *Smack*
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Badass goodlooking piece of a hot woman: Fuck off! *Smack*
Dude: *wraf*
por Fessus Fessheim 09 de Agosto de 2006
Trending Now
Can you define these popular missing words?
Japanese school girl uniform popular in cosplay.
You are WAY to old to go in a fuku.
por Ash1231231231232123 19 de Fevereiro de 2011
A sarcastic way to say "fuck you". Mostly used on chat services such as IRC and other sites that have chats. It's not meant to be taken seriously, so keep this in mind.
Person 1: Wow this is so stupid
Person 2: fuk u
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Person 1: no u
por LucasRCD 31 de Julho de 2016
An informal way to say "goodbye and good luck with whatever you're trying to accomplish" when you're so flabbergasted by an individual's or group's words or actions that you only have 4 letters in you. Not at all related to profanity or the work "fuck"
Person: Donald Trump would make a great president.
Logical human: fuk u
Logical human: fuk u
por RT4T 02 de Janeiro de 2016
Can typically start any internet rebuttal (There are exceptions, usually based on common sense) and sometimes ends with 'Also, Penis.' for an attempted humorous twist.
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internatsdood23: fuk u.
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por Raggs 31 de Julho de 2008
A way to say, "Fuck You" to a higher authority without getting in trouble.
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President Obama Visits Baton Rouge After Criticism That He's Late
President Obama tours Louisiana's communities affected by the flood that damaged over 60,000 homes and killed 13 people.
Wayne Bradley, the new state director of African-American engagement in Michigan. (Photo: Courtesy of Michigan Republican Party)
Republican National Committee chairman Reince Priebus met with a group of African-American business and community leaders in Detroit on Tuesday to discuss its ongoing black voter outreach initiative and ways to revitalize struggling urban centers.
Priebus announced that radio host Wayne Bradley will serve as state director of African-American engagement effort and launched a 13-member Michigan Black Advisory Council to assist him and build relationships throughout the state.
"We are committed to continuing our engagement efforts in the Black community and will not stop competing for every vote, year-round. Our engagement staff and advisory board bring tremendous value to the work the Republican Party hopes to accomplish," the chairman said in a news release.
He told MLive.com that the meeting was "just the beginning" and that he plans to implement a block-by-block outreach approach in the state.
Bradley is a Detroit native and founder of the Urban Conservative League, a community-based organization that advocates school choice and private school voucher programs.
"I am thrilled to engage with local leaders and members of my community as I share the positive vision of the Republican Party with them," he said.
BET Politics - Your source for the latest news, photos and videos illuminating key issues and personalities in African-American political life, plus commentary from some of our liveliest voices. Click here to subscribe to our newsletter.
President Obama tours Louisiana's communities affected by the flood that damaged over 60,000 homes and killed 13 people.
The only person that can send Gatlin home without the gold medal? A young man from Trelawney, Jamaica, whose last name literally means to run.


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Display advertising has made major leaps over the past few years; today, it's a tool tailor-made for direct response marketers with a programmatic mindset. Unfortunately, many direct response marketers – haunted by memories of display's days as a low-click crapshoot – remain reluctant to embrace modern display advertising. Simply put, they don't know what performance display looks like. It's time to debunk those myths.
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Right…and social media can only be used for contests. Display is much more than a pretty banner that makes people know that a brand exists. Every day, more performance marketers are using display advertising to drive conversions through precisely targeted ads based on a user's site and search behavior. There's a misconception that display is on its way out the door. In fact, it's growing faster than SEM.
Myth No. 2: Display Is Expensive
This is true when marketers go directly to sites to buy placements – where they can end up paying up to a $100 CPM – or when they insanely spend $450,000 for a one-day Yahoo home page takeover.
These tactics are unnecessary and downright foolish. The vast majority of publishers have a significant amount of remnant inventory, which is made available through auctions on ad exchanges. The average CPM for remnant inventory is between $1 and $2. Even in super competitive verticals such as finance, display can often be a cheaper (and more efficient) acquisition channel than SEM.
Myth No. 3: Display Doesn't Work
You know what doesn't work? Click metrics. Clicks seemed like a novel way to measure display advertising when they first burst onto the scene, but they really had no connection to the way that advertising actually works. After all, print ads, TV ads, and billboards have been working for a long time now without anyone actually clicking on them. Want a much more accurate measurement? View-through attribution. View-through attribution – the practice of measuring users who convert a short time after seeing a display ad – just makes more sense. Though view-through attribution lost some credibility when marketers gamed it by setting insanely long conversion windows (like 30 days), we're seeing that, when measured correctly, view-through makes much more sense than counting clicks.
Myth No. 4: Display Ads Don't Talk to the Right People
Display has undergone a dramatic transformation in the past year, as search retargeting has emerged as one of the most effective forms of intent-driven advertising that we've ever seen. When you combine the power of search marketing with the advanced precision of programmatic site retargeting, you have a combination that allows you to find exactly the right people and deliver exactly the right message.
Myth No. 5: Display Advertising Is Not Exciting
Display advertising is advancing at a rapid pace: the hyper-evolution of ad exchanges, dynamic creative, retargeting pixels, and display inventory has made it one of the most complex forms of advertising. That may not be exciting to some people, but, at least for data nerds like me, it can be a lot of fun.
If you don't believe it, just look at Facebook's newest development, where users can be retargeted with socially activated ads right in their news feeds, meaning that you can share great content with the right people in the place they're most likely to see it. And if that doesn't get you excited, you might be in the wrong business.
Image on home page via Shutterstock.
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