Genetically Manipulated Food News

13 January 99

Table of Contents

UK restaurants press for ban on GE crops
Guardian wins complaint ruling
Group opposes cow hormone
Fears of growth hormone in milk
More on Aromatic Amines? Tryptamine may transform into carcinogenics
Monsanto Announces Agreement on Glyphosate and Roundup Ready Crops
Complex array of label rules gives US exporters headaches
Old-fashioned corn to resist super bugs
CNN.com Launches Food Central
Time Magazine articles on biotech
A Virus Promoter Used in the Majority of Genetically Engineered Crops
35S Promoter (CaMV) in Calgene's Flavr Savr Tomato Creates Hazard
Research Abstracts re Cauliflower Mosaic Virus
Publications about CaMV
Spread Of Type I Introns In Plants
Evolution: Explosive Invasion Of Plant Mitochondria By A Group I Intron

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Date: 11 Jan 1999 00:15:15 -0600
From: Judy_Kew@greenbuilder.com (Judy Kew)

UK restaurants press for ban on GE crops

By Paul Brown
The Guardian Weekly Volume 160 Issue 2 for week ending January 10, 1999, Page 11

THE country's most prestigious restaurants are calling on the Government to impose a five-year ban on growing and selling genetically modified food while further research on its impact is carried out.

Celebrated chefs such as Nico Ladenis, of Chez Nico at Ninety Park Lane, London, and Raymond Blanc, of Le Manoir aux Quat' Saisons in Great Milton, Oxfordshire, have joined a campaign calling for the ban.

Chez Nico, which has three Michelin stars, leads a list of the best restaurants which regard genetically modified food with distaste. Only 23 restaurants in Britain rate 8 out of 10 or more in the 1999 Good Food Guide for their "quality of cooking". Nineteen of these back the Friends of the Earth campaign for a five-year ban on genetically modified food.

The restaurants are asking for the five-year ban because the results of field trials on genetically modified crops will not be known before then. The trials start this year to measure the spread of herbicide-resistant crops into the environment, for example to see whether they inter-breed with weed species or "normal" plants and/or reduce insect and bird populations.

The Government intends to permit the release of genetically modified crops for commercial use in 2000 -- four years before the results of its safety checks are known.

Adrian Bebb, of Friends of the Earth, said: "By the time the Government has found out whether there are adverse affects it will be too late to do anything about it. We are tampering with our food, creating extra risk, yet there is no demand or benefit except for the multinationals that market the seed and the herbicides needed to go with it."

The Commons catering committee, which runs all the canteens and restaurants in the House of Commons, has banned genetically modified food.

A Mori survey this year found that 61 per cent of the public did not want genetically modified food and 77 per cent supported a ban on commercial growing of these crops.

Matthew Townsend
Barrister & Accredited Mediator
Lecturer in Environmental Law, Victoria University of Technology
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Date: 11 Jan 1999 03:34:32 -0600
From: jim@niall7.demon.co.uk (jim mcnulty)
Publication Date: January 10, 1999

Guardian wins complaint ruling

The Guardian

© Copyright 1999, _____via IntellX_____

THE Press Complaints Commission yesterday rejected complaints from the US biotech company, Monsanto , against the Guardian.

Monsanto had complained about "inaccuracies and misleading statements" in an analysis piece which examined the growing opposition to the introduction of genetically modified foods in Britain.

Yesterday the commission said: "After review, the commission has decided that no matters have been raised which show a breach of the code."

Monsanto had objected to five points in an analysis article published in July 1998, which was accompanied by a map that depicted some of the sites where the company had been testing modified crops.

The Guardian's managing editor, Brian Whitaker, said yesterday: "We are delighted by the ruling. The complaint was typical of the response whenever we write about Monsanto in the context of genetically-modified foods.

"We are well accustomed to vigorous lobbying from public relations companies, but Monsanto seems to put enormous resources into complaining every time we write about its activities."

The commission ruled that consumer and pressure groups had expressed anger over a lack of consultation by Monsanto; that Monsanto had been unsuccessful in its attempts to restrain protesters; and that the Guardian could not be criticised for inaccuracies in a map drawn up from government information.

The commission declined to rule on the Guardian's request that it consider Monsanto's reasons for making the complaint.

It said: "It was not for the commission to comment on the complainant's motivation."


Date: 11 Jan 1999 03:34:48 -0600
From: jim@niall7.demon.co.uk (jim mcnulty)

Group opposes cow hormone

The Patriot Ledger Quincy, MA
© Copyright 1999, _____via IntellX_____

WASHINGTON -- An advocacy group said Monsanto Co. 's Posilac, the synthetic hormone used to boost milk production, should be suspended from the U.S. market and regulators should hold further hearings on its safety. The Washington-based Center for Food Safety said it's petitioning the U.S. Food and Drug Administration to review the agency's 1993 approval of the hormone after Canadian drug regulators raised questions about its effect on rats. "Their concerns are groundless," said Monsanto spokesman Gary Barton. "There's no new science."


Date: 11 Jan 1999 03:35:47 -0600
From: jim@niall7.demon.co.uk (jim mcnulty)

Fears of growth hormone in milk

South China Morning Post
© Copyright 1999, _____via IntellX_____

What could be less controversial than milk? In Canada, as it turns out, a lot.

Consumer activists are sounding alarm bells and government scientists are accusing their bosses of bending to the powerful chemical industry.

The uproar concerns a proposal by chemical giant Monsanto to allow farmers to use a growth stimulant in feed for cows in order to boost milk production.

Farmers in the United States and some other countries already use bovine somatotropin, a hormone that raises milk production in cows by 10 to 15 per cent, but Canada has been much more cautious in approving its use.

That caution was manifested by six health protection branch scientists in Ottawa, who gained national prominence after complaining that their superiors pressured them to approve the drug despite their concerns that it might not be safe.

Monsanto, which has been pushing for approval for eight years, says it is perfectly safe for both humans and cows and is in big demand by dairy farmers.

A labour tribunal dismissed the complaints of the government scientists that they faced undue workplace pressure but it also said it "had no authority to examine and assess the scientific concerns raised".

The debate came to a head during parliamentary hearings in Ottawa. A Monsanto representative complained that the company's reputation had been unfairly tarnished and warned that its experience with the Health Department might discourage other companies from doing business in Canada.

Nevertheless, officials say the department will not decide whether to permit use of the hormone until the summer at the earliest - when a United Nations food safety committee is expected to discuss the drug - and have talked about asking Monsanto for long-term health studies.


Date: 11 Jan 1999 10:58:27 -0600
From: Jaan Suurkula m-25430@mailbox.swipnet.se

More on Aromatic Amines? Tryptamine may transform into carcinogenics

At 15:27 1999-01-05 in a former answer to Mark Gold I wrote (clipped):

Mark: 1. Are they saying that aromatic amine levels increase in the plants they are talking about?

Jaan: Yes, the (aromatic amine) tryptamine levels were increased in a specific strain of transgenic tobacco. This had a pesticidal effect by making pests sterile.

2. Mark. Would a similar increase be possible in other transgenic crops?

Jaan: If it is desired, it might occur. But every gene insertion is unique, so it is impossible to say how proable it would be. But in this case, the tryptamine increase seems to have been accidental. As it seems to be a major cause for the pesticidal effect it was considered a possible new solution for controlling pests. Provided that this "unexpected metabolic effect" could be reproduced in other cases of gene insertion.

3. Mark: Are these the same type of risky aromatic amines referred to in the article below?

Jaan: It is not specified in the article what Aromatic Amines (AAS) were found to be the cause of cancer. But AAS is a large class of chemicals and some are known carcinogens. Tryptamin is not as far as I know a carcinogen.

==============================================================

I have looked further for info on tryptamin. And I found an article reporting that tryptamin can be transformed into a carcinogen by endogenous enzymes (endogenous means produced naturally within own body). The enzymes in question were acetyltransferases (responsible for acetylation of the nitrogen and the oxygen of arylamine derivatives).

This was taken to explain (rather technical text - you may want to jump to the Conclusion below) "the occasional induction of tumors in organs not usually considered as targets of aromatic amines, as well as raise the possibility that the production of N-oxidized endogenous substrates may represent a mechanism for tumor induction in the absence of exogenous carcinogens."

CONCLUSION

The possiblity of transformatin of tryptamin into carcinogens by own enzymes, might have the consequence that in food organisms where tryptamine has been increased to abnormal levels by GE, there might be an increased risk for the appearance of oxidized tryptamin derivates that are carcinogenic. With the present inadequate GE food safety assesment method applying "Substantial Equivalence" there is a significant risk that such substances would pass undetected (see http://www.psagef.org/subeqow.htm. This was found in animals. It takes a biologist to judge to what extent a similar biochemical possibility exists in plants. Considering that tryptamine exists in plants, and that the the biochemistry of plants and animals is similar, this seems conceivable.

Reference:

TITLE:Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines.
AUTHORS:King CM; Land SJ; Jones RF; Debiec-Rychter M; Lee MS; Wang CY
AUTHOR AFFILIATION:Carcinogenesis Program, Karmanos Cancer Institute, Detroit, MI, USA.
SOURCE:Mutat Res; VOL 376, ISS 1-2, 1997, P123-8 (REF: 23)

An abstract of this reference can be found at the Toxline database: http://igm.nlm.nih.gov Search words: tryptamine+carcinogen

Jaan Suurkula MD Chairman of PSRAST

_______________________________________________________________
PHYSICIANS AND SCIENTISTS FOR RESPONSIBLE APPLICATION OF
SCIENCE AND TECHNOLOGY(PSRAST)
- A global network -

E-mail: psrast@swipnet.se Tel: +46-322622966 Fax: +46-322620944
Website: http://www.psagef.org/index.htm


Date: 12 Jan 1999 03:53:37 -0600
From: jim@niall7.demon.co.uk (jim mcnulty)

Monsanto Announces Agreement on Glyphosate and Roundup Ready Crops

SOURCE: Monsanto Company, Company Press Release, Monday January 11, 8:55 pm Eastern Time

ST. LOUIS, Jan. 11 /PRNewswire/ -- Monsanto Company (NYSE:MCT - news) announced today a U.S. licensing agreement with Nufarm Ltd. that allows Nufarm to register its own brand of glyphosate herbicide over the top of certain Roundup Ready crops.

Under the new agreement, Nufarm will reference for a fee Monsanto's registration data and will be authorized to sell its own brand of glyphosate over the top of Roundup Ready soybeans and Roundup Ready cotton beginning October 1, l999 and over Roundup Ready corn beginning October 1, 2000 in the United States.

Monsanto and Nufarm announced previously a number of glyphosate herbicide supply agreements that expanded the geographic coverage globally from previous supply agreements for glyphosate and provided access for a fee to Monsanto glyphosate registration data in certain countries. Monsanto and Nufarm's agreement is consistent with our policy to provide said Patrick In addition, the licensees acknowledge and respect the intellectual property and registration data associated with the technology -- and have committed to

Monsanto's Roundup and Roundup Ultra herbicides, which include the active ingredient glyphosate, and Nufarm's glyphosate brand, will compete with each other in the agricultural marketplace.

All performance, crop safety and stewardship support concerning all uses of Nufarm's brand of glyphosate, including over the top of pertinent Roundup Ready crops, is the responsibility of Nufarm.

No additional details were provided and all other terms are confidential.

As a life sciences company, Monsanto is committed to finding solutions to the growing global needs for food and health by sharing common forms of science and technology among agriculture, nutrition and health. The company's 30,000 employees worldwide make and market high-value agricultural products, pharmaceuticals and food ingredients.

This press release contains certain forward-looking statements, including those related to the market for and sales of Roundup Ultra herbicide. These forward-looking statements are based on past experience and current expectations, but actual results may differ materially from those anticipated and there can be no guarantee that future results will be similar to those of the past. Certain factors which could cause actual results to differ materially from expected and historical results include: weather; price; new use; patent expiration; local farming practices; local economic conditions; the type of crops planted; and the availability, price and desirability of competitive products; as well as economic, competitive, governmental, intellectual property, technological and other factors identified in Monsanto Company's Form 10-K and Form 10-Q filings with the Securities and Exchange Commissio


Date: 12 Jan 1999 14:05:27 -0600
From: jim@niall7.demon.co.uk (jim mcnulty)

No different to the complex array of people at this end refusing to eat it, I think. Poor supermarkets and poor exporters, if only they had listened and segregated it from the beginning, we would have seen the end of it before now I reckon. Jim Mc Nulty.

Complex array of label rules gives US exporters headaches

Journal of Commerce, Publication Date: January 11, 1999

When the Euro-bureaucrats in Brussels order new labels, they can stick it to a host of exporters across the Atlantic Ocean.

Whether those label requirements concern genetically modified organisms (GMO) or metric-only measurements, such rules cause headaches for U.S. exporters who must comply so they can compete in European markets.

When those labeling regulations are unclear, or the rules are inconsistently enforced, exporter headaches can turn into migraines.

"It's very frustrating for an exporter to find out about a new labeling requirement and then not be able to tell whether his product complies," said a U.S. official familiar with the GMO-labeling rule. "With this GMO rule, nobody really knows how to come into compliance."

As far as labeling goes, that GMO regulation is now the brittlest bone of contention between U.S. exporters and European Union officials.

In recent years, U.S. farmers have been planting an increasing number of acres with soybeans and corn -- such as Monsanto Corp. 's "Roundup-Ready" soybeans -- that are genetically modified to help fight weeds or insect pests.

Because the altered beans or corn look the same as "normal" ones, it is difficult to segregate the modified crops during harvesting, storage and transport. That makes it tough for U.S. food makers who use such crops to certify that they do or do not contain genetically modified ingredients.

Last spring, the EU decided to require labeling of food products containing genetically modified ingredients. The reason: Even though there is no scientific evidence that GMOs pose health risks, Europeans are generally more skeptical than Americans about altered foods, and they want labels to show clearly whether some food-product ingredients are derived from GMOs.

The problem is that the new "novel foods" labeling regulation, which went into effect Sept. 3, does not specify a "minimum threshold" -- below which no GMO labeling would be required -- or a specific criteria for testing for GMOs.

Also, officials in Brussels have not yet issued a list of products -- such as highly refined soya oil, which shows no sign of genetically modified proteins -- that are not subject to GMO labeling.

"How can you tell if you are in compliance with the labeling law if they haven't set a minimum level of genetically modified ingredients allowed, and they haven't approved a testing method?" asked a U.S. agriculture official.

The holes in the regulation also are confusing European food makers, especially those that use U.S.-grown soybeans or corn. "Clearly, there are problems," said Dominiqe Taeymans, an expert at the Brussels-based European Confederation of Food and Drink Industries. "We have asked the European Commission to act to clarify the situation."

Jochen Kubosch, a spokesman for the commission, says "there are technical problems" with the GMO-label rules, which the EU will try to correct, and that EU experts are working on a list of food products to be exempt from the GMO rule. "There should be such a list, but for the time being it is empty," he said.

Meanwhile, U.S. companies have been scrambling to try to comply with the EU labeling requirements.

In one approach, Protein Technologies International Inc. -- a DuPont Corp. subsidiary that markets soy proteins and fibers for use mainly in food products -- went to the expense of developing an "Identity Preservation System" that tracks soybeans through every step from the farms where they are grown to the silos that store them to the trucks that transport them.

"Developing this system was very expensive," said Kathy Harris, the St. Louis-based company's general counsel. "But it now allows us to assure our European customers that the soy protein we sell them is not derived from genetically modified material -- and they don't have to use the "Contains GMO' label that the EU is requiring." Protein Technologies sells its soy products to a wide range of European food- making companies, which the company says accounts for a significant portion of its $466 million in annual sales. Like many other U.S.- based firms, it maintains a Brussels-based subsidiary that keeps track of various EU regulations affecting its business.

But genetic modification is not the only labeling issue affecting U.S. companies.

A decade ago, the EU insisted that all product labels list metric- system weights and measures, such as grams -- and not dual labels that list both metric and U.S./British equivalents, such as ounces.

The U.S. government has managed to stall the metric-only directive, but unless the EU issues another extension, it will go into effect as of Jan. 1, 2000.That would cause major problems for many U.S. exporters. In October, the Trans-Atlantic Business Dialogue -- representing leading industries from both sides of the Atlantic -- urged the European Commission to delay implementation of metric-only labeling for another 10 years "to prevent imposing additional costs on industry and to eliminate regulatory uncertainty." The U.S. government has taken the same position.

More than 80 U.S. companies -- including such major exporters as 3M, DuPont, IBM Corp. and Mobil Corp. -- and several trade associations joined a coalition to lobby EU governments to postpone the law indefinitely.

But EU officials seem to be leaning more toward an extension of from three to five years. Said Mr. Kubosch, the commission spokesman, "We are working on an extension now, which should be ready for consideration in the next two months. But I can't say how long that extension would last."

© Copyright 1999, _____via IntellX_____


Date: 12 Jan 1999 14:06:03 -0600
From: jim@niall7.demon.co.uk (jim mcnulty)

Well folks, I have seen it all now, old fashion corn, like my grandpapi used to grow. These people are so stuck up their backsides, they can't see the light of day. If it wasn't a sick joke it might have rated alongside, the story that I found after it. Please read. Jim Mc Nulty.

Could always nuke the super bugs I suppose.

Old-fashioned corn to resist super bugs

Chicago Sun Times

A coalition of the nation's major producers of genetically engineered corn seed announced Friday that they would require farmers to grow sizable plots of non-engineered, old-fashioned corn along with their new varieties. The companies hope to allay increasing fears among scientists that some newly marketed varieties of gene-altered corn, which exude potent insecticides day in and day out, may be speeding the evolution of pesticide-resistant "super" insects.

The announcement was made by Monsanto , the No. 1 producer of genetically altered corn seed, at a meeting of the Environmental Protection Agency. U.S. cultists kicked out of Israel Members of a U.S. cult, suspected by Israel of plotting violence in Jerusalem to bring about the second coming of Christ, were deported early today. Fourteen cultists, including six children, took off for Denver where the group is based.

There is growing concern in Israel that the group, the Concerned Christians, is a forerunner of hundreds of fanatics who will be drawn to Israel at the close of the millennium for what they expect to be the return of Jesus. Israeli officials fear that such extremists could turn violent if their hopes are not met, and some predict mass suicides at holy sites. The suburban Jerusalem homes of the 14 cultists were raided earlier this week, and the 14 were detained. They denied wrongdoing, and no charges were filed.

© Copyright 1999, _____via IntellX_____


Date: 12 Jan 1999 17:53:52 -0600
From: jtoth@lisco.com (Joe Toth)

CNN.com Launches Food Central

Offering Up-to-the-minute Food and Nutrition News and Information

Company Press Release, Monday January 11, 12:47 pm Eastern Time

Digital Chef is Site's Premier Recipe Provider with Direct Links to Shopping for Thousands of Gourmet Food and Kitchenware Items

ATLANTA--(BUSINESS WIRE)--Jan. 11, 1999-- CNN.com launches FOOD CENTRAL, the newest addition to its heavily trafficked feature sections. Located at CNN.com/Food, Food Central offers the latest food and nutrition news together with recipes, restaurant reviews and other useful resources for food-lovers around the world. It also hosts more than 75 message boards, allowing Internet users to swap recipes and tips, as well as review their favorite restaurants. Digital Chef, the largest online gourmet food and kitchenware retailer and the online cooking authority, provides FOOD CENTRAL daily recipe specials and illustrated step-by-step guides to help users perfect their cooking techniques. CNN.com's Food Central allows users to keep up-to-speed on all the latest issues affecting food and diet and at the same time form online communities to share restaurant and cooking said Scott Woelfel, editor in chief and vice This is yet another example of how CNN can be the one online source for all kinds of news and Digital Chef works closely with The Culinary Institute of America, assuring that users are getting the most authoritative cooking information on the Internet when they access Digital said We are delighted to be the featured provider of daily recipes and cooking tips on CNN's new food section and look forward to showing CNN.com's millions of users that they can easily shop for gourmet foods, kitchenware and other cooking related

FOOD CENTRAL, which is linked to related content in the health, travel and books sections of CNN.com, features:

Recipes-a week's worth of Digital Chef's daily specials, including a servings calculator and nutritional information, a recipe finder that searches thousands of exclusive recipes from Digital Chef, healthy alternative recipes from Mayo Clinic Health Oasis and links to other recipe Web sites;

News-the top reports from the world of food and nutrition with interactive polls, related links and related stories;

Restaurants-reports from CNN Travel Now, CNN's new international travel and food show, and restaurant reviews from more than 50 U.S. and worldwide cities from the Weissmann Travel Report, a leading provider of destination information to the travel industry;

Discussion-cooks and gourmets can swap recipes on specialized message boards covering everything from low fat to Thai cooking, post restaurant reviews as well as get tips and offer advice to other Food Central patrons; -Digital Chef offers illustrated step-by-step guides teaching trade secrets from some the country's best known chefs and CNN provides basic 101 cooking tips.

Resources-information on a range of dietary and nutritional topics, including nutrition comparisons of hundreds of foods, with links to other food-related Web sites.

Digital Chef is the largest online retailer of gourmet food and kitchenware offering thousands of gourmet foods, professional quality cooking tools and brand name kitchen appliances like Kitchen Aid and Cuisinart. A joint venture with The Culinary Institute of America (CIA), the nation's leading center for culinary education, Digital Chef is the online authority on cooking and provides thousands of recipes from the CIA, cooking instruction from some of the world's best-known chefs, and online shopping for the best foods, kitchenware and cooking tools for every occasion.

CNN Interactive is the division of CNN that produces CNN.com, the award-winning general news and information site, as well as five other Web sites and other major digital news products and services. The CNN Web sites are the most visited online destination for news and information, generating more traffic and attracting more unduplicated Internet users than any other company's general news and information Web sites.

Contact:

CNN Interactive
Kerrin Roberts, 404/827-5340, kerrin.roberts@turner.com or
Digital Chef
Margaret Ryan, 415/243-9243, margaret@digitalchef.com


Date: 12 Jan 1999 18:59:27 -0600
From: jtoth@lisco.com (Joe Toth)

Time Magazine articles on biotech

Time Magazine this week has a complete set of mostly PR, spin doctoring, Big Money appealing articles:

THE FUTURE OF MEDICINE
JANUARY 11, 1999
"The Biotech Century"
http://cgi.pathfinder.com/time/magazine/articles/0,3266,17687,00.html
The food stuff is at:
Brave New Farm
The first commercial products bred by genetic engineering incur a backlash in Europe, where "Frankenstein" fears run deep By JAMES WALSH/LONDON"
http://cgi.pathfinder.com/time/magazine/articles/0,3266,17698,00.html

The site has a WRITE TO US link at the bottom...

-=-=-=-=-=-=-=-=-=-=-=

Please sign the Consumer Right to Know Petition : Just send the following message to ge-mail@lofthouse.com

"Add my name to the Consumer Right to Know Petition for the mandatory labeling of genetically engineered foods."

Make sure to include your name and address in USA (which will only be used for this petition). For more info, hard copies of the petition and for friends without email to sign, call toll-free: 1 877 REAL FOOD (1 877 732 5366) or visit the Mothers for Natural Law web site: http://www.safe-food.org/-campaign/petition.html


Date: 13 Jan 1999 04:30:21 -0600
From: akb@akb.a2000.nl

Here are a number of articles on CaMV promoter. Some are very technical.

A Virus Promoter Used in the Majority of Genetically Engineered Crops

Prof. Joe Cummins
Professor Emeritus of Genetics
University of Western Ontario, 738 Wilkins Street, London, Ontario N6C4Z9 Canada
e-mail: jcummins@julian.uwo.ca

Sections:
The Cauliflower Mosaic Virus: CaMV
References
The Use Of Cauliflower Mosaic Virus
Looking for CaMV-literature in PubMed

The Cauliflower Mosaic Virus: CaMV

In crop genetic engineering genes from various sources including plants, bacteria or even mammals including humans are placed in crop plants. For those genes to function a regulator gene called a promoter is essential. The promoter turns the introduced gene on and maintains its output at a high level. Promoter genes of the crop plant do not work very well with foreign genes so a powerful promoter that controls replication of a virus has been used extensively because it functions in a most plant species from corn to pine trees. The Cauliflower Mosaic Virus (CaMV) promoter has been used in virtually all of the genetically engineered plants.

The CaMV promoter proved to be a key solution to the problem of keeping foreign genes active in crop plants. It is not only effective in gene activation but it is also compact and easy to manipulate, it is readily available at low cost from gene suppliers. The CaMV promoter took over most promoter applications in crop biotechnology because it gives consistent high level expression of foreign genes in crops and no other promoter whether from virus or plant has been found to supersede it. However, use of the gene poses threats to the environment and to human health and discussion of these threats have been ignored, for the most part, in regulatory reviews and risk analysis.

CaMV is a pararetrovirus, closely related to the human Hepitis B virus and less closely related to retroviruses including HIV the cause of AIDS(1). Retroviruses and pararetroviruses share the trait of alternating their genes between RNA and DNA and reverse transcribing the RNA to make DNA. The virus promoter is a small gene that acts to start replication of the DNA into many virus copies. When that gene is attached to a gene from a bacterium, crop plant or animal then added to a crop plant chromosome many copies of the foreign gene are produced by the plant. The promoter gene is active in all the cells of the crop plant and little influenced by environmental conditions or the crop cellAEs genetic controls.

The CaMV promoter is active in most plant species from simple vascular plants to the giant evergreen trees. It is also active in yeast and insect cells as well(2,3). The CaMV virus is transmitted by insects,pollen and by contact between plants. Even though the virus promoter is active in plants, yeast and insect cells the virus is spread through a limited number of plant families, but these include a number of important crops both crucifers and crops such as potatoes and beans(4).

The major threat from the CaMV promoter in crops is the process of recombination. Numerous studies showed that CaMV genes genetically engineered into crop plants recombine with infecting viruses to create viruses that are unique and frequently much more infectious than the infecting virus (5,6,7,8,9). These recombination events are fully capable of creating viruses that spread in an uncontrolled manner and cause food shortages and famine worldwide. Such recombination events have been estimated to take place at one per million cell division(10), a moderately sized plant would have over a thousand such recombinats while a large plant or tree could have a million recombinants.

Furthermore, CaMV promoter has been associated with clones that are DNA copies of a wide range of viruses, such clones produced infectious virus (11,12) . Recombination leading to clones that would be carriers of potent infecting viruses is a real possibility. The CaMV promoter is a ohot spoto for genetic recombination(13). That promoter is the starting point for replication of the virus, most recombination appears to start at the promoter gene. Recombination may take place when RNA virus is reverse transcribed to make DNA or when the

DNA form of the virus makes RNA, in the latter case the recombinant RNA is returned to the DNA form by reverse transcription. The normal replication cycle of the virus is that the virus is spread in its DNA form. When the virus infects the plant cell it first replicates as RNA, which is then reverse transcribed to make DNA. Recombination takes place at both the RNA and the DNA phase of replication. The CaMV promoter gene in the chromosome and its neighboring genes participate in recombination to make recombinant virus. As indicated earlier, such recombinant viruses may be more virulent than the original virus.

The ability of the CaMV promoter to create steady elevated level of gene product can lead to problems. For example, lectins are natural plant defense toxins used to defend against browsing from predators and disease causing pests. Such lectins are present at low level in many crops but when elevated using CaMV promoter may exceed levels toxic to the immune system of mammals. Lectins, such as snowdrop lectin extensively field tested and patented may not be safe at the level achieved in genetically engineered crops. Wheat germ and barley lectins are well known to be toxic but are being introduced into a number of crop plants.

Field testing has allowed the spread of such genes in the environment before the safety of the constructions has been proved. When lectin modified crops are marketed they are unlikely to be labeled , in that way jeopardizing sensitive people such as the celiac population sensitive to wheat gluten and associated lectin. Field tests(14) and patents(15) for lectin genes in crops have been taking place for the past seven years while the public have been left unaware of the hazard. CaMV promoter has been used to activate a number of mammalian genes in crop plants, the gene products are mainly vaccines, dietary proteins or disease treatments. There has been little thought given to the hazard of recombinant forms of such genes. One interesting construction involved use of a mammalian interferon gene in crops to fight crop virus. The genetically engineered tobacco plants resisted one virus completely but all the plants died immediately when infected by a different virus, a result completely unexpected (16). Unpredictable toxicity of genetically engineered crops is a worrisome reality.

CaMV DNA has been used in disturbing experiments with infants and children in a day care setting. The DNA was used to trace spread of pathogens in the day care setting (17). Such experiments are unsafe because the DNA may be taken up through skin lesions or breathed attached to dust particles. Such DNA may penetrate cells and produce products that are allergens or toxins.

In conclusion, CaMV genes are used in most of the genetically engineered crops. The impact of these genes has not been effectively evaluated. Experiments with infants and children do not contribute effectively to the safety evaluation of the many genetic constructions with CaMV. Such experiments should not have been permitted by an effective ethical review process.

References

  1. Xiong,Y. and Eikbush,T. Origin and evolution of retroelements based upon the reverse transcriptase sequences. The EMBO Journal 9,3363-62,1990.

  2. Smerdon,G.,Aves,S. and Walton,E. Production of human gastric lipase in the fission yeast Gene 165,313-8,1995.

  3. Vlack,J.,Scoulten,A.,Usmany,M., Belsham,G., KlingeRoode,E.,Maule,A.,VanLent,M. and Zuidema,D. Expression of Cauliflower Mosaic Virus Gene 1 Virology 179,312-20, 1990.

  4. Brunt,A.,Crabtree,K.,Dallwitz,M.,Gibbs,A.,Watson,L. and Zurcher ,E. (eds.) Plant Virus Online Version:16th January 1997 URL: http://biology.anu.edu.au/Groups/MES/vide

  5. Schoez,J. and Wintermantel,W. Expansion of viral host range through complementation and recombination in transgenic plants The Plant Cell 5,1669-79,1993.

  6. Gal,S.,Pisan,B.,Hohn,T.,Grimsley,N. and Hohn,B. Genomic homologous recombination in plants EMBO Journal 10,1571-78,1991.

  7. Gal,S.,Pisan,B.,Hohn,T.,Grimsley,N. and Hohn, B. Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intramolecualr recombination Virology 187,525-33,1992.

  8. Gal,S.,Pisan,B.,Hohn,T.,Grimsley,N. and Hohn, B. Agroinfection of transgenic plants leads toi viable cauliflower mosaic virus by intramolecular recombination Virology 187,525-33,1992.

  9. Wintermantel,W. and Schoeelz,J. Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure Virology 223,156-64,1996

  10. Swoboda,P.,Hohn,B. and Gal,S. Somatic homologous recombination in plants Mol.Gen.Genet 237,33-40,1993.

  11. Maiss,E.,Timpe,U.,Brisske-Rode,D,Lesemann,E. and Caper,R. Infection in vitro transcrips od plum pox potyvirus in full length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter J. Gen. Virol. 73,709-13,1992.

  12. Meyer,M. and Dessens,J. 35S promoter driven cDNAs of barley mild mosaic virus are infectious on barley plants J.Gen. Virolo 78,147-51,1997.

  13. Vaden,V. and Melcher,U. Recombination sites in cauliflower mosaic virus DNAs Virology 177,717-26,1990.

  14. ICGEB Biosafety Webpage http://base.icgeb.trieste.it:80/biosafety/bsfrei.htm

  15. Patents on lectin genes:

  16. Ogawa,T.,Hori,T. and Ishida,I. Virus induced cell death in plants expressing the mammalian 2AE,3AE oligoadenylate system Nat. Biotechnology 14,1566-9,1996.

  17. Jiang,X.,Goldblatt,S.,Buescher,C.,Cusak,T.,Matson,D. and Pickering.L. Pathogen transmission in child care setting studied by using a cauliflower virus DNA J.Infect. Dis. 177,881-8,1998

The Use Of Cauliflower Mosaic Virus

35S Promoter (CaMV) in Calgene's Flavr Savr Tomato Creates Hazard

Joseph E. Cummins
Professor Emeritus (Genetics) Dept. of Plant Sciences University of Western Ontario, June 3, 1994

The majority of crop plant constructions for herbicide or disease resistance employ a Promoter from cauliflower mosaic virus (CaMV). Regardless of the gene transferred, all transfers require a promoter, which is like a motor driving production of the genes' message. Without a promoter, the gene is inactive, but replicated, CaMV is used because it is a powerful motor which drives replication of the retrovirus and is active in both angiosperms and gymnosperms. The CaMV pararetrovirus replication cycle involves production vegetative virus containing RNA which is reverse transcribed to make DNA similar to HIV, Human Leukemia Virus and Human hepatitis B. (Bonneville et al. RNA Genetics Vo.11, Retroviruses, Viroids and RNA Recombination pp. 23-42, 1988). CaMV is closely related to hepatitis B and is closely related to HIV (Doolittle et al. Quart.Rev.Biol. 64,2, 1989; Xiong and Eickbush, EMBO Joumal 9, 3353, 1990).

The CaMV promoter is preferred above other potential promoters because it is a more powerful promoter than others and is not greatly influenced by environmental conditions or tissue types. CaMV has two Promoters 19S and 35S, of these two the 35S promoter is most frequently used in biotechnology because it is most powerful. The 35S promoter is a DNA (or RNA) sequence about 400 base pairs in length. The use of the CaMV promoter in plants is analogous to the use of retrovirus LTR promoters in retrovirus vectors used in human gene therapy. The majority of human gene therapy trials employ LTR promoters to provide motors to activate genes.

Antisense genes are genes constructed to have a complementary sequence to a target gene, thus producing a product that combines with a gene message to inactivate it. Antisense is analogous to an antibody which combines with an antigen like a key fitting a lock. Antisense is being used to treat human cancer and HIV infection. Antisense is used to prevent spoilage in tomatos, either by targeting an enzyme degrading cell walls (polygalacturonase), or production of ethylene a hormone promoting ripening (P. Oeller et al. Genetic Engineering 49, 1989; R. Fray and D. Grierson, Trends Genetics 9, 438, 1993). Most frequently antisense targets production of a chemical metabolite producing ethylene. The antisense gene also influenced polyamines spermine and spermidine production through S-adenosylmethionine. The implication is that the plant antisense gene product should be tested in animals to ensure that critical functions including gene replication, sperm activity and gene imprinting are not disrupted.

The perceived hazards of CaMV in crop plants include the consequences of recombination and pseudo recombination. Recombination is the exchanges of parts of genes or blocks of genes between chromosomes.

Pseudorecombination is a situation in which gene components of one virus are exchanged with the protein coats of another. Frequently viruses may incorporate cellular genes by recombination or pseudorecombination, it has been noted that such recombinants have selective advantages (Lai, Micro. Rev. 56, 61, 1992).

It has been shown that the CaMV genes incorporated into the plant (canola) chromosome recombine with infecting virus to produce more virulent new virus diseases. The designers of the experiment questioned the safety of transgenic plants containing viral genes (S. Gal et al., Virology 187: 525, 1992). Recombination between CaMV viruses involves the promoter (Vaden and Melcher, Virology 177: 717, 1992) and may take place either between DNA and DNA or RNA and RNA and frequently creates more severe Infections than either parent (Mol. Plant-Microbe Interactions 5, 48, 1992).

Recently related experiments suggest altered plants may breed deadlier diseases (A. Green and R. Allison, Sciences 263: 1423, 1994). DNA copies of RNA Viruses are frequently propagated using the CaMV 35S promoter to drive RNA virus production (J.Boyer and A. Haenni, Virology 198: 4l5, 1994 and J.Desuns and G.Lomonossoff, J. Gen. Vir. 74: 889, 1993). In conclusion CaMV promoters recombine with the infecting viruses to produce virulent new diseases. CaMV viruses and promoter may incorporate genes from the host creating virulent new diseases.

CaMV can recombine with insect viruses and propagated in insect cells (D. Zuidema et al. J. Gen. Vir. 71: 312, 1990). Thus it is likely that as large numbers of humans consume CaMV modified tomatos recombination between CaMV and hepatitis B viruses will take place creating a supervirus propagated in plants, insects and humans.

Plant biotechnology has grown out of recombinant DNA research that began in the early 1970's. The special nature of recombination has been debated since that time. In recent years, government regulators on the American and European continents, under pressure from well-funded lobby representing the biotechnology industry, have chosen to ignore the special nature of recombination. They have chosen instead to base regulations on existing frameworks for toxic chemicals and pathogenic organisms. Ignoring the special nature of recombination is likely to have costly, if not terminal, environmental consequences. A worst-case example includes the complete cloning of Human Immunodeficiency Virus (HIV) on an E. coli plasmid. When the plasmid is used to transform animal cells, intact HIV viruses are released from the cells.

A careless (but legal) release of HIV bacteria to the environment would allow the plasmid to transfer to Salmonella as well as E. coli. Thus, numerous mammals and birds could contain HIV bacteria which could transform the animals, which would in turn produce HIV particles unable to target the animals T-cell receptors but easily transmitted to humans. When all the animals are HIV carriers, human survival would be marginal. The special concerns of recombination in plant biotechnology include the viruses and bacteria used in crop plant construction and gene flow between related crop plants and weeds in the field.

Currently most experts agree that virus diseases such as influenza gain strength for epidemics by alternating between animal hosts (pigs and ducks) and man. Epidemics begin when rare combinations appear in large closely associated populations such as in asia. CaMV can propagate in plant and insect hosts following recombination. It may not be outlandish to predict that CaMV may recombine with related Hepatitis B or for that matter HIV to create a most powerful disease. The salient feature being large number of people or animals consuming large numbers of virus genes incorporated into crop plants making up a major part of human and animal diet.

The use of CaMV promoter is seldom an issue in reviews of safety of gene tinkered crops. Few people have raised the important issue and more often than not their concerns are ignored by government officials "protecting" public safety. This omission may be a fatal one because it has potentially the most damaging impact, and the one perceived at the beginning of gene splicing. (The FlavrSavr tomato brand has been withdrawn because it turned out to have unexpected deficiencies that did not make it useful for commercial production.)

Looking for CaMV-literature in PubMed

http://www.ncbi.nlm.nih.gov/PubMed and give in the search 'CaMV'

Some results:


Research Abstracts re Cauliflower Mosaic Virus

  1. Virus Genes 1998;17(2):139-50 The cauliflower mosaic virus capsid protein: assembly and nucleic acid binding in vitro.

    Chapdelaine Y, Hohn T Friedrich-Miescher Institute, Basel, Switzerland.

    [Medline record in process]

    The capsid protein of the cauliflower mosaic virus (CaMV) was expressed in a bacterial system to study CaMV assembly. Bacterial lysates contained soluble particulate material and insoluble inclusion bodies that were both used for analysis. In vitro renaturation of pIV derivatives lead to the appearance of folded sheets or large tubular structures in electron microscopy. The region between amino acid positions 77 and 332 is sufficient for self-aggregation of pIV in vitro. C-terminal deletion to amino acid position 265 still allowed dimerization but prevented further aggregation. Nucleic acid binding assays of immobilized pIV derivatives demonstrated that a region located upstream of the retroviral "zinc finger-like" motif is involved in unspecific binding dsDNA, ssDNA and RNA. PMID: 9857987, UI: 99073698

  2. Plant Mol Biol 1998 Dec;38(6):1053-60 Gibberellin and abscisic acid regulate GAST1 expression at the level of transcription.

    Shi L, Olszewski NE Department of Plant Biology, University of Minnesota, St. Paul 55108, USA.

    [Medline record in process]

    Both gibberellic acid (GA3) and abscisic acid (ABA) regulate the expression of the GAST1 gene of tomato. Treatment with GA3 increases the abundance of GAST1 RNA while treatment with ABA blocks this effect. In this study, the effects of GA3 and ABA on the rate of transcription of the GAST1 gene and the stability of GAST1 RNA were examined.

    Nuclear run-on analyses detected an increase in transcription of the GAST1 gene 1 h after GA3 treatment with transcription increasing to a maximum at 9 h after treatment. The half-life of GAST1 RNA in GA3-treated leaves was similar to that in control leaves. In addition, the extent of overexpression of GAST1 RNA in transgenic tomato plants containing the CaMV 35S promoter driving the expression of the GAST1 transcribed region was largely unaffected by GA3. These results suggest that GA3 stimulates the expression of the GAST1 gene by acting only at the level of transcription. ABA treatment dramatically reduced the abundance of GAST1 RNA in gib1 shoots through an effect at the level of transcription and did not appear to affect the stability of this RNA.

    Midcourse ABA addition to the GA3-incubated shoots reversed the GA3-mediated increase in the transcription of GAST1 gene within 15 min. Transgenic plants that either overexpressed or underexpressed GAST1 RNA exhibited no phenotypic differences from wild type. PMID: 9869411, UI: 99084756

  3. Microbiol Immunol 1998;42(5):377-86 Requirement of cauliflower mosaic virus open reading frame VI product for viral gene expression and multiplication in turnip protoplasts.

    Kobayashi K, Tsuge S, Nakayashiki H, Mise K, Furusawa I Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University, Japan. kappei@basic.kpu-m.ac.jp

    Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6.

    This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, IV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended on the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication. PMID: 9654370, UI: 98316942

  4. Microbiol Immunol 1998;42(4):329-34 Evidence for a dual strategy in the expression of cauliflower mosaic virus open reading frames I and IV.

    Kobayashi K, Tsuge S, Nakayashiki H, Mise K, Furusawa I Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University, Japan. kappei@basic.kpu-m.ac.jp

    Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-beta-glucuronidase (GUS) fusion constructs.

    In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes. PMID: 9623922, UI: 98285403

  5. Genetics 1998 Jun;149(2):693-701 Characterization of the putative transposase mRNA of Tag1, which is ubiquitously expressed in Arabidopsis and can be induced by Agrobacterium-mediated transformation with dTag1 DNA.

    Liu D, Crawford NM, Department of Biology, University of California at San Diego, La Jolla, California 92093, USA.

    Tag1 is an autonomous transposable element of Arabidopsis thaliana. Tag1 expression was examined in two ecotypes of Arabidopsis (Columbia and No-0) that were transformed with CaMV 35S-Tag1-GUS DNA. These ecotypes contain no endogenous Tag1 elements. A major 2.3-kb and several minor transcripts were detected in all major organs of the plants. The major transcript encoded a putative transposase of 84.2 kD with two nuclear localization signal sequences and a region conserved among transposases of the Ac or hAT family of elements.

    The abundance of Tag1 transcripts varied among transgenic lines and did not correlate with somatic excision frequency or germinal reversion rates, suggesting that factors other than transcript levels control Tag1 excision activity. In untransformed plants of the Landsberg ecotype, which contain two endogenous Tag1 elements, no Tag1 transcripts were detected. Agrobacterium-mediated transformation of these Landsberg plants with a defective 1.4-kb Tag1 element resulted in the appearance of full-length Tag1 transcripts from the endogenous elements. Transformation with control DNA containing no Tag1 sequences did not activate endogenous Tag1 expression. These results indicate that Agrobacterium-mediated transformation with dTag1 can activate the expression of Tag1. PMID: 9611184, UI: 98278791

  6. Plant Mol Biol 1998 Jun;37(3):581-5 A plant transformation vector with a minimal T-DNA II. Irregular integration patterns of the T-DNA in the plant genome.

    Porsch P, Jahnke A, During K
    Federal Centre for Breeding Research on Cultivated Plants, Institute for Breeding Methods in Vegetables, Quedlinburg, Germany.

    A minimal T-DNA binary vector was used for Agrobacterium-mediated transfer of a chimeric T4 lysozyme gene located next to the left border, and transgenic potato plants which expressed T4 lysozyme protein were identified and further analysed. Frequent rearrangements of T4 lysozyme transgenes were detected. A vector derivative containing two matrix associated regions (MARs) flanking its multiple cloning site was constructed. In transgenic potato plants, reduced variability in gene expression due to position effects was detected.

    When either the donor vector contained MAR sequences, or when vector pPCV701 which contains a pBR322 fragment next to the left border were used, only relatively few rearrangements were observed. However, when the T4 lysozyme gene was driven by a CaMV 35S promoter modified by multiplied enhancer region carrying either 2 or 4 elements, frequent rearrangements were again obtained. PMID: 9617825, UI: 98278387

  7. Plant Mol Biol 1998 May;37(2):287-96 The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent.

    Haldrup A, Petersen SG, Okkels FT
    Danisco Biotechnology, Copenhagen K, Denmark.

    The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation.

    The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the omega' translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection. PMID: 9617801, UI: 98278363

  8. Biochem Biophys Res Commun 1998 Apr 17;245(2):403-6 Methylation of nonintegrated multiple copy DNA in plants.

    Tang W, Leisner S
    Biology Department, University of Toledo, Ohio 43606, USA.

    DNA sequences present in multiple copies in plant genomes are often methylated. However, it was not known if methylation occurs on multiple copy DNA molecules that are not integrated into the plant genome. To investigate this possibility, the methylation state of cauliflower mosaic virus [CaMV] DNA was studied in inoculated turnip leaves at different days post-inoculation [DPI]. Unencapsidated CaMV DNA was found in an unmethylated state up to 7 DPI. By 9 DPI, viral DNA was methylated at almost all HpaII/MspI sites within the CaMV genome. DNA methylation did not appear to be preferential but occurred at almost all HpaII/MspI sites at approximately the same time. Methylation appeared to occur in an all or none manner, suggestive of a switch mechanism. These data strongly suggest that copy number-dependent methylation occurs on DNA molecules that are not integrated into the genome of a host cell. PMID: 9571163, UI: 98238649

  9. Microbiol Immunol 1998;42(1):65-9 Accumulation kinetics of viral gene products in cauliflower mosaic virus-infected turnip protoplasts.

    Kobayashi K, Nakayashiki H, Tsuge S, Mise K, Furusawa I
    Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University, Japan. kappei@basic.kpu-m.ac.jp

    The expression of cauliflower mosaic virus (CaMV) genes was studied in a turnip protoplast system. Six CaMV-encoded gene products were detected in infected turnip protoplasts by means of Western blotting. The infected turnip protoplasts showed different patterns of protein accumulation; e.g. an open reading frame (ORF) I-encoded movement protein, an ORF V-encoded reverse transcriptase and an ORF VI-encoded posttranscriptional transactivator representing the early accumulated proteins, an ORF II-encoded aphid transmission factor and an ORF IV-encoded coat protein the late accumulated proteins and an ORF III-encoded DNA binding protein the intermediate protein. The results suggest that the expression of CaMV genes is differentially regulated. PMID: 9525783, UI: 98184697

  10. Virology 1998 Mar 15;242(2):403-13 Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA in transgenic tobacco plants.

    Scharer-Hernandez N, Hohn T
    Friedrich Miescher-Institut, Basel, Switzerland.

    Cauliflower mosaic virus (CaMV) uses a specialised translation mechanism to bypass the long leader sequence of the 35S RNA. The effect of the CaMV 35S RNA leader sequence on the expression of a downstream beta-glucuronidase (GUS) reporter gene was studied in transgenic tobacco plants. Enzymatic GUS assays of these transgenic plants show that a shunt mechanism of translation indeed occurs in planta with an average efficiency of 5% compared with the leaderless construct. Histological GUS analyses indicate that the shunt mechanism occurs throughout the whole plant and at all developmental stages. PMID: 9514980, UI: 98190360

  11. Virology 1998 Mar 15;242(2):395-402 Mapping regions of the cauliflower mosaic virus ORF III product required for infectivity.

    Jacquot E, Geldreich A, Keller M, Yot P
    Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique, Universite Louis Pasteur, Strasbourg, France.

    The open reading frame (ORF) III product (PIII) of the pararetrovirus cauliflower mosaic virus (CaMV) has nucleic acid-binding properties in vitro, but its biological role is not yet determined. ORF III is closely linked to ORF II and overlaps ORF IV out of frame in the CaMV genome. A new CaMV-derived vector (Ca delta) devoid of ORF III and containing unique restriction sites between ORFs II and IV was designed. Introduction of the wild-type CaMV ORF III into Ca delta results in a clone (Ca3) infectious in turnip plants. Truncated or point-mutated versions of ORF III were then inserted into Ca delta and tested in vivo.

    Inoculation of the different mutants into turnip revealed that the four C-terminal amino acid residues of PIII are dispensable for infectivity as well as an internal domain (amino acids 61 to 80). Taken together the results show that PIII possesses a functional two-domain organization. Moreover, the CaMV PIII function(s) cannot be replaced either by the PIII protein of another caulimovirus, the figwort mosaic virus, or by the P2 protein of the cacao swollen shoot badnavirus, a member of the second plant pararetrovirus group. PMID: 9514961, UI: 98190359

  12. From an Internet Home-page of Friedrich Miescher Institute (FMI) Mattenstrasse 66, CH 4058 Basel (Switzerland) secretary@fmi.ch Molecular plant virology

    T. Hohn Thomas.Hohn@fmi.ch, M. Bliffeld*, Y. Chapdelaine, G. Chen, S. Corsten, D. Dominguez, XY. He, M. Hemmings-Mieszczak, N. Hernandez, A. Himmelbach, Z. KissLaszlo*, D. Leclerc, A. Lee*, D. Lewetag, J.-L. Mougeot, M. Muller, H.Rothnie, L. Ryabova*, W. Schmidt-Puchta* (*left since the last Report)

    Introduction

    Viruses serve as excellent models for vari ous aspects of molecular biology. Genome replication, transcriptional and translation control and assembly have been and still are studied successfully using viral model sys tems. The components of viruses can further be used to study the rules of protein- and RNA folding and of protein-protein and protein-nucleic acid interactions. We are working with two plant pararetroviruses: cauliflower mosaic virus (CaMV) infecting dicots and rice tungro bacilliform virus (RTBV) infecting monocots. This choice allows comparative studies, e.g. how the two viruses master their common problem to translate several cistrons from one RNA. Deeper understanding of virus regulation might lead to new antiviral strategies, while more of their components could be exploited as toolboxes for biotechnology (5,7,8).

  13. CaMV transcriptional control: methylation and silencing

    S. Corsten, M. Muller, H. Rothnie

    A methylation-target free (MTF) derivative of the CaMV 35S promoter was constructed. Treatment with SssI-methylase, which methylates CG motifs, still inactivated MTF-GUS reporter genes. Mosaic methylation experiments showed that this effect is based on methylation of the coding region and that it seems to be stronger if regions close to the5'-end are methylated. Introduction of a MTF leader and intron diminished the nega tive effect of coding region methylation. Transgenic plants containing the MTF promoter-GUS construct expressed GUS well in leaves but poorly in roots.

    In collabo ration with other groups, we are testing whether some types of silencing can be pre vented by use of MTF constructs. CaMV translational control: structure of the leader and ribosome shunt (W. Schmidt-Puchta, D. Dominguez, D. Lewetag, M. Hemmings-Mieszczak) The pregenomic RNAs of CaMV and RTBV have a 600-nt-long leader sequence harbor ing a number of small open reading frames and potentially forming a large hairpin struc ture. This piece of RNA contains several regions involved in various aspects of virus replication. While the leader structure princi pally inhibits translation, a special shunt mechanism transporting the scanning ribo somes from a position close to the cap-site to upstream of the first ORF counteracts this inhibition.

    The CaMV leader RNA was studied by chemical and enzymatic probing and also by temperature gradient electrophoresis, allow ing isomorphs and their melting behavior to be analyzed simultaneously. Our studies ver ified the large stem-loop structure covering most of the leader, and revealed additional structural elements, i.e. three pseudoknots. Two of these (e1 and e2) could replace part of the large stem, the third one (e3) connects the central and 3'-terminal portion of the leader in a long-range interaction. RNAs containing e3 could be distinguished by their slower mobility and by their in creased stability at high ionic strength and low temperature. Mutants were designed to stabilize or destabilize the pseudoknot iso morphs. Stabilization of either e1 or e3 led to an increase in translation efficiency.

    UV cross linking identified two turnip cytoplasmic proteins, p49 and p100, that bind specifically to regions of the CaMV leader. Interestingly the two strongest inter actions occurred within regions overlapping e1 and e2.

    Conditions were found to optimize cap-dependence and minimize backgrounds of internal initiation in the wheat germ transla tion system. This allowed us to study shunt ing in vitro and to definitively exclude any nontranslational process as the cause of the shunting phenomenon. In this system oligonucleotides in antisense to the 5' and 3' portions of the leader (including the e1 and e2 regions) inhibit translation initiation, while oligonucleotides in antisense to the central portion of the leader do not.

  14. CaMV translation control: transactivation and transactivator protein

    A. Himmelbach, N. Hernandez

    The CaMV gene VI product is a multi functional protein interacting with RNA, viral proteins and the translational machin ery. In transiently transfected protoplasts and in transgenic plants it allows translation of both ORFs of dicistronic RNAs. In trans genic plants, gene VI caused chlorotic symp toms and much delayed flowering. Sucrose gradient centrifugation experiments revealed the association of pVI with mono- and poly somes and far-western experiments identi fied an 18S ribosome- or ribosome associ ated plant protein as the interaction target (6).

  15. Splicing in CaMV

    Z. Kiss-Laszlo

    Transient expression experiments using re porter constructs revealed a splice donor site within the leader of CaMV 35S RNA and three additional sites within ORF I. All four donors use the same splice acceptor within ORF II. The corresponding spliced CaMV RNAs were detected in infected plants. Vi rus mutants of the splice acceptor were not infectious unless reversions occurred. Splic ing between the leader and ORF II produced an mRNA from which ORF III and, in the presence of the CaMV translational trans activator, ORF IV could be efficiently trans lated (9,10).

  16. Polyadenylation

    H. Rothnie

    Examination of the sequences surrounding the RTBV poly(A) site has revealed many similarities to the CaMV poly(A) signal. An essential AAUAAA motif lies just upstream of the processing site, and U-rich sequences further upstream are required to enhance processing efficiency. Evidence so far sug gests that poly(A) site regulation involves a mechanism distinct from those described for other retroelements. The poly(A) site is effi cient, is not inhibited by promoter proxim ity, and no sequences upstream of the tran scription start site are required. In constructs where the RTBV leader was present directly downstream of either the CaMV or the RTBV promoter, the majority of the tran scripts were processed at the poly(A) sitewithin the leader, giving rise to high levels of "short-stop" RNA. However, this RNA was barely detectable in infected plants, sug gesting that a virus function or cis-element actively represses the promoter proximal poly(A) signal.

  17. Properties of CaMV structural proteins

    Y. Chapdelaine, M. Hemmings-Mieszczak, A. Himmelbach, D. Leclerc, J-L. Mougeot

    The CaMV pre-capsid polyprotein (pIV) is thought to interact with a variety of viral-and host proteins. The yeast two-hybrid sys tem and far-western experiments revealed interactions of the capsid protein with itself and with the viral proteins pIII (minor cap sid component) and pVI (inclusion body protein and transactivator). The site interact ing with pVI was mapped to the C-terminal basic cluster. The capsid protein region in volved in nucleic acid binding mapped to the same domain (6). CaMV pIV is extensively processed at both ends during virus assembly. Three tar gets for phosphorylation by the virus-associ ated kinase were identified near the N-termi nus of p44, one of the derivatives of pIV. Comparison of phosphorylation profiles and reactivity of antibodies raised against the C-terminal Zn-finger motif indicated that p39 is derived from p44 by N-terminal trunca tion and p37 from p39 by C-terminal trunca tion.

    The capsid proteins contain N-terminal and C-terminal clusters of basic amino ac ids. After transient expression in plant protoplasts, capsid protein derivatives con taining the N-terminal RKRK motif, but not a RARA mutation of it, were transported to the nucleus. The C-terminal basic clusterwas not as efficient as an NLS, but in con nection with the N-terminal motif directed accumulation of the capsid protein to a subnuclear domain, probably the nucleolus.

  18. Expression strategies of rice tungro bacilliform virus

    G. Chen, X-Y. He, M. Muller and H. Rothnie, in collaboration with J. Futterer, A. Klothi and I. Potrykus, ETH, Zurich and R. Hull, John Innes Institute, Norwich

    The RTBV DNA sequence upstream of the transcription initiation site has only very weak promoter activity (2). However, at least two distinct components could be iden tified downstream of the initiation site, i.e. within the leader region, which acted to gether to strongly enhance transcription (1). One of these elements is a position- and orientation-independent DNA element, while the second is position-dependent. Nu clear proteins interacted with the first DNA element and with a transcript of the second one. A related DNA-based element was also found in the CaMV leader.

    The first ORF following the leader (ORF I) lacks an ATG initiation codon. Initiation-competent ribosomes are delivered very pre cisely to the second of two potential ATT initiation codons by a process similar to the ribosome shunt mechanism observed in CaMV. ORFs II and III are reached by leaky scanning by those ribosomes that fail to ini tiate at ORF I. ORF IV is translated from a spliced RNA (4).

  19. Proteins and RNAs involved in translational control of tobacco mosaic virus

    A. Lee, L. Ryabova

    The omega sequence and trailer of tobacco mosaic virus cooperate in enhancing transla tion in a wheat germ system. UV-cross link ing identified several proteins binding both to the leader and the trailer region but not to other RNAs. One of these was identified as eIF4a by its ATP- dependence and by its reaction with specific antibodies. Two other proteins, p26 and p28, are tentatively as signed to eIF4F and eIF-(iso)4F due to their molecular weights and purification behavior and because the cap and TMV trailer com pete for their binding. p26 and p28 bind to the pseudoknot region of the TMV trailer but not to the t-RNA region. They also bind to the CaMV RNA terminal repeats, but not to the trailers of some other viruses ( e.g. PVX). These results indicate a role in some virus trailers to recruit translation initiation factors.


Publications about CaMV

  1. Chen G (1995) Regulation of gene expression in rice tungro bacilliform virus: Involvement of leader based enhancer elements. PhD thesis, University of Basel

  2. Chen G, Muller M, Potrykus I, Hohn T, Futterer J (1994) Rice tungro bacilliform virus: transcription and translation in protoplasts. Virology 204: 91-100

  3. Filipowicz W, Gniadkowski M, Klahre U, Liu H-X, Hemmings-Mieszczak M (1994) Pre-mRNA splicing in higher plants. In: de Kouchkovsky Y, Larher F (eds) Plant sciences 1994. SFPV - University of Rennes I

  4. Futterer J, Potrykus I, Valles-Brau MP, Dasgupta I, Hull R, Hohn T (1994) Splicing in a plant pararetrovirus. Virology 198: 663-670

  5. Goldbach R, Hohn T (1995) Plant viruses as gene vectors. In: Bryant JA (ed) Methods in plant molecu lar biology. Academic Press, Boston (in press)

  6. Himmelbach A (1995) Cauliflower mosaic virus ORF VI protein. Interactions with the translational machinery and the ORF IV protein. PhD thesis, University of Basel

  7. Hohn T (1994) Recombination of plant pararetroviruses. In: Paszkowski J (ed) Homologous recombi nation and gene silencing in plants. Kluwer Academic, Dordrecht, pp 25-38

  8. Hohn T, Goldbach R (1994) Vectors: plant viruses. In: Webster RG, Granoff A (eds) Encyclopedia of virology, vol 3. Academic Press, London, pp 1536-1543

  9. Kiss-Laszlo Z (1995) Expression of the downstream ORFs from the polycistronic 35S mRNA of cauli flower mosaic virus. PhD thesis, University of Basel

  10. Kiss-Laszlo Z, Blanc S, Hohn T (1995) Splicing of Cauliflower Mosaic Virus 35S RNA is essential for viral Infectivity. EMBO J 14: 3552-3562

  11. Klahre U, Hemmings-Mieszczak M, Filipowicz W (1994) Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA binding proteins in Nicotiana plumbaginifolia. Plant Mol Biol 28: 569-574

  12. Rothnie HM, Chapdelaine Y, Hohn T (1994) Pararetroviruses and retroviruses: a comparative re view of viral structure and gene expression strate gies. Adv Virus Res 44: 1-67

  13. Rothnie HM, Reid J, Hohn T (1994) The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3'-end forma tion in plants. EMBO J 13: 2200-2210

  14. Simpson GG, Clark GP, Rothnie HM, Boelens W, van Venrooij W, Brown JWS (1995) Molecular characterisation of the spliceosomal proteins U1A and U2B" from higher plants. EMBO J 14: 4540-4550

  15. Wera S, Fernandez A, Lamb N, Turowski P, Hemmings-Mieszczak M, Mayer-Jaekel R, Hemmings B (1995) Deregulation of translational control of the 65-kDa regulatory subunit (PR 65*) of protein phosphatase 2A leads to multinucleated cells. J Biol Chem 270: 21374-21381

FMI WebMaster: Carl David Nager, Friedrich Miescher Institute (FMI) Mattenstrasse 66, CH 4058 Basel (Switzerland) Last update: March 20, 1996

email (for reprints,...) to secretary@fmi.ch

-----Ursprüngliche Nachricht-----
Von: AKB Amsterdam [SMTP: akb@xs4all.nl]
Gesendet am: Freitag, 08. Janner 1999 11:51
Betreff: Re: Dr pusztai


Date: 13 Jan 1999 04:33:06 -0600
From: akb@akb.a2000.nl Subject: [Fwd: Introns]

AKB Amsterdam wrote:

Hi joe,

Thanks for the mail. I hope you are fine by now and not too disturbed by storms and strikes. I have a question on introns. I assume you heard about the study that introns are moving with great speed into a large number of plantspecies. From what I was reading about it it is Intron 1. I do not know about the classification in introns and what this means. I am wondering however to which extent this is a dangerous development.

Do you anything about this subject?

Best wishes

wytze

Spread Of Type I Introns In Plants

Joseph E. Cummins
Professor Emeritus (Genetics) Dept. of Plant Sciences University of Western Ontario,

I am enclosing an article on the spread of type I introns in plants. The article is from the Proc. Natnl. Acad. Sci. USA . Type I introns are spliced from messenger RNA, they are self splicing, meaning they do not need enzymes to be spliced.

Self splicing depends on very short gene sequences that are very mobile, they are being used in medical genetic engineering to treat cancer, AIDS, etc. Such introns are usually restricted to the mitochondria of plants and fungi. Human and other "higher" mammals don't have introns in their mitochondrial chromosomes. Infection of human mitochondrial chromosomes with such sequences could have profound consequences, damage to mitochondria genes leads to a number of hereditary diseases transmitted through maternal lines, these include blindness, retardation and a number of related conditions.

Damage to mitochondrial chromosomes most rapidly and profoundly effects brain functions because the brain is very very sensitive to changes in oxidative metabolism.

Joe Cummins

------------------

joe cummins wrote:

Hi Wytze,

Thanks for the question. I know introns, I taught advanced molecular genetics for thirty years and the discoverers of introns are all my friends. Introns are fundamental , they arte are genes within genes. The dangerous, and they are very dangerous introns have the ability cut and splice genes without using enzymes.sometimes they create a problem called dysgenesis that leads to steriltiy or weakened reproduction and birth defects in populations. I will forward my letters on this to Maiwan, to you.

Cheers,

Joe


Date: 13 Jan 1999 04:33:27 -0600
From: akb@akb.a2000.nl

joe cummins wrote: Hi Wytze, Here is the Proc. Natnl. Acad. article I negelected pasting :-( Cheers, Joe

Vol. 95, Issue 24, 14244-14249, November 24, 1998

Evolution: Explosive Invasion Of Plant Mitochondria By A Group I Intron

Yangrae Cho, Yin-Long Qiu*, Peter Kuhlman, and Jeffrey D. Palmer
Department of Biology, Indiana University, Bloomington, IN 47405

Communicated by Susan R. Wessler, University of Georgia, Athens, GA, September 24, 1998

(received for review July 28, 1998)

Group I introns are mobile, self-splicing genetic elements found principally in organellar genomes and nuclear rRNA genes. The only group I intron known from mitochondrial genomes of vascular plants is located in the cox1 gene of Peperomia, where it is thought to have been recently acquired by lateral transfer from a fungal donor. Southern-blot surveys of 335 diverse genera of land plants now show that this intron is in fact widespread among angiosperm cox1 genes, but with an exceptionally patchy phylogenetic distribution.

Four lines of evidencethe intron's highly disjunct distribution, many incongruencies between intron and organismal phylogenies, and two sources of evidence from exonic coconversion tractslead us to conclude that the 48 angiosperm genera found to contain this cox1 intron acquired it by 32 separate horizontal transfer events. Extrapolating to the over 13,500 genera of angiosperms, we estimate that this intron has invaded cox1 genes by cross-species horizontal transfer over 1,000 times during angiosperm evolution. This massive wave of lateral transfers is of entirely recent occurrence, perhaps triggered by some key shift in the intron's invasiveness within angiosperms.

* Present address: Institute of Systematic Botany, University of Zürich, Zollikerstrasse 107, 8008 Zürich, Switzerland.

Present address: Department of Chemistry and Biochemistry, Denison University, Granville, OH 43023.

To whom reprint requests should be addressed. e-mail: jpalmer@bio.indiana.edu.

Copyright © 1998 by The National Academy of Sciences 0027-8424/98/9514244-6$2.00/0

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