A Time & A Place for Gene Transfer

It is late in the afternoon, so here’s some sex to spice up my talk. (slide 1) These two butterflies, poised so delicately on a flower, are engaged in rampant gene transfer of the natural kind. And they probably stay like that for hours, quite remarkable considering their short lifespan. I was snapping shots with my digital camera at every conceivable angle for at least half an hour until I got tired.

Those butterflies know that there is a proper time and place for gene transfer, and they only do it with soul mates.

Life originated some 3.8 billion years ago, when the earth’s atmosphere was a poisonous mixture of carbon monoxide, carbon dioxide, methane, hydrogen, hydrogen sulphide, and even cyanide, and the only living things that could have survived were bacteria that could transform those common chemicals to obtain energy, like the heat-loving bacteria or thermophiles that still exist today around volcanic vents, mud holes and hot seeps at great depths on the ocean floor. Archaebacteria like these are thought to have been the first life forms, although no trace of them has been preserved in our rocks. Here’a a picture from Yellowstone Park, the vivid colours on the ground are the thermoacidophile bacteria, which not only love boiling heat but also strong sulphuric acid. (slide 2)

The oldest fossils, stromatolites, were found in South African chert, dating to 3.5 billion years before present. (slide 3) These remarkable compact structures are layers of photosynthetic cyanobacteria mats overlying other microbes. The cyanobacteria traps sunlight to split carbon dioxide into carbon to build the bacteria, releasing oxygen, eventually changing the atmosphere to support organisms that, like us, need oxygen. Some stromatolites still survive today, in Western Australia, for example, where they grow quite big. (slide 4)

The first Eukaryotes - organisms with genetic material enclosed in a nucleus - did not appear until 1.6 billion years ago, in the form of green algae. What about the single celled animals? They left no trace. According to current theory, plants, animals and fungi were all derived from simpler ‘protists’ rather like the amoeba. So a plant is just an amoeba that has eaten a chloroplast and suffers a permanent indigestion.

The oldest multicellular animal fossils were a weird and wonderful collection, first discovered in the Ediacara Hills of the Flinders Mountains in south Australia (slide 5). Dickinsonia, like a corrugated pita loaf, Spriggina, with crescent-shaped head and a segmented body, Cyclomedusa, now thought to be the stalk of a sedentary jelly-fish, Tribrachidium, with three loopy arms, and Parvancorina, a heart-shaped beast with a gut that branches into two. The fossils date to 600 million years before present, in the late preCambrian era, and animals like these are thought to have originated a billion years ago. It took 2.5 billion years of photosynthesis, therefore, to sufficiently enrich the early atmosphere with oxygen for these animals to evolve.

The next fossil fauna to appear in geological time are the early Cambrian animals, first discovered in the Burgess Shale, high in the Canadian Rockies of British Columbia, in the Yoho National Park on the flanks of Mount Stephen. (slide 6) Stephen Jay Gould featured them in his book, Wonderful Life, and made much of the profusion of unusual body plans among them. Unlike the Ediacaran fossils, many had shells, similar to arthropods, molluscs and trilobites. Although they still look strange, the majority eventually found places within existing phyla of invertebrates.

The world changed dramatically between the late preCambrian and the Cambrian, within about 15 million years at the most, but could be much shorter, as animals like those of the Burgess Shale were found among the earliest rocks of the Cambrian. The Cambrian-Precambrian boundary is now placed at 545 mya. The appearance of exoskeleton or shells was part of the transition, but not the whole story, according to Richard Fortey in Life, an Unauthorised Biography. He believes that the ancestors of the arthropods, molluscs and all the rest were already there in the late preCambrian, but were very small, so they couldn’t readily be preserved as fossils, and only left their record after the conditions of earth made it possible for them both to acquire shells and to become larger.

The rest of evolution is relatively familiar history. The geological epochs are defined by origins, major transitions, radiations and extinctions. The first vertebrates appeared 500 million years ago, the amphibians and insects 405 mya, and extensive forests of early vascular plants already covered the earth by 345 mya. At 280 mya, the continents aggregated into Pangaea, a gigantic landmass, accompanied by marine extinction and radiation of reptiles. Dinosaurs became abundant in the Jurassic around 181mya and suffered mass extinction together with other marine and terrestrial life 135mya. The radiation of mammals and birds, insects and angiosperms took place in the Tertiary, beginning 63 mya, and the evolution of primates, and the Homo species followed in the last few million years before present.

There is a time and place for every major phyla and every species to come on the evolutionary stage. The species did not all evolve at once, or all in the same place. Gene transfer was restricted within species boundaries, although there were exceptions especially among bacteria, where genetic material had been transferred horizontally between unrelated species, and far back towards the beginnings of life, horizontal gene transfer occurred between the ancestors of the three major domains of Archaea, Bacteria and Eukaryotes. As a rule however, organisms, including bacteria, have many ways to prevent foreign DNA getting into their genomes.

That is important, particularly in view of discoveries that geneticists have been making since the mid 1970s, that tell us there is really no holding the genome still. Francis Crick and James Watson may be largely right about the structure of DNA, but they are woefully wrong on how genes are supposed to determine the characteristics of organisms in linear, one-way causal chains, rather in the manner of an ‘unmoved mover’.

Francis Crick’s infamous Central Dogma of molecular biology decrees that genetic information flows strictly one way, from DNA to RNA to protein, and by implication, to the characteristic determined by that protein.(slide 7) That must have been the inspiration for genetic modification. And if it were really that simple, genetic modification would work just fine.

But even as biotech companies were set up, genetic engineering as a research tool was making momentous discoveries that would overturn every single assumption of genetic determinism and eventually sink the industry. A more representative picture is what geneticists in the early 1980s were already calling, "the fluid genome". (slide 8)

Gabriel Dover and Dick Flavell, two geneticists who played key roles in defining the new genetics, put it succinctly in 1982: (slide 9)

"The application of new molecular techniques reveals that, beneath the level of the chromosome, the genome is a continuously changing population of sequences. Mobility, amplification, deletion, inversion, exchange and conversion of sequences create this unexpected fluidity on both an evolutionary and developmental timescale."

More recently, James Shapiro has referred to the same processes as the "natural genetic engineering" that the organism has to do to survive. But this natural genetic engineering has all the appearance of being quite precise, and to be regulated by the organism as a whole. The fluidity of the genome reflects the constant intercommunication that has to take place between all parts of the organism to enable it to make a good living from its ecological environment.

Genetic modification therefore, is breaking all the rules of evolution. Genetically modified organisms are unnatural, not just because they have been produced in the laboratory, but because many of them can only be made in the laboratory, quite unlike what nature has produced in the course of billions of years of evolution. It is short-circuiting the evolutionary process.

It involves recombining or joining together new combinations of DNA from widely different sources, and deliberately inserting the artificial constructs into the genomes of organisms.

Thus, it is possible to introduce new genes and gene products, from bacteria, viruses and other species, or even genes made entirely in the laboratory, into crops, including food crops, that we have never eaten; that have never even been part of our food chain. Here is a typical unit construct. (slide 10) A gene is never transferred alone, it needs a promoter, a gene switch to say to the cell "copy the following message for making a protein", and another signal to say, "stop here, end of message". All three parts are often from different sources. The gene itself could also be a composite of different DNA.

The artificial constructs are further spliced into gene carriers and introduced into cells by invasive methods that result in random integration into the genome, giving rise to unpredictable, random effects, including gross abnormalities in animals and unexpected toxins and allergens in food crops. (slide 11) In other words, there is no possibility for quality control. This problem is compounded by the overwhelming instability of transgenic lines, because the artificial constructs cobbled together from DNA of different sources tend to have weak joints.

Since September 11, 2001, the world has been whipped up into hysteria over terrorist attacks and ‘weapons of mass destruction’. Governments want to ban publication of sensitive scientific research results, and a group of major life sciences editors and authors has concurred. Some scientists even suggest an international body to police research and publication. But few have acknowledged that genetic engineering itself is inherently dangerous.

What caught the attention of the mainstream media in January 2001 was how researchers in Australia ‘accidentally’ created a deadly mouse virus that killed all its victims in the course of manipulating a harmless virus. (slide 12) "Disaster in the making: An engineered mouse virus leaves us one step away from the ultimate bioweapon", was the headline in the New Scientist article. The editorial showed even less restraint: "The genie is out, biotech has just sprung a nasty surprise. Next time, it could be catastrophic."

That and the current SARS epidemic (slide 13) serve to remind us it is horizontal gene transfer and recombination that creates new viruses and bacteria that cause disease epidemics, and if genetic engineering does anything, it is to greatly enhance the scope and tendency for horizontal gene transfer and recombination.

First, genetic engineering allows the rampant recombination of genetic material from widely diverse sources that would otherwise have very little opportunity to mix and recombine in nature. Some newer techniques, as for example, ‘DNA shuffling’ will create in the matter of minutes millions of new recombinants in the laboratory that have never existed in billions of years of evolution. There is no limit to the sources of DNA that can be shuffled in this way, including DNA that is now found to be retrievable from fossils 300 000 to 400 000 years old.

Second, disease-causing viruses and bacteria and their genetic material are the predominant materials and tools of genetic engineering, as much as for the intentional creation of bio-weapons. And this includes antibiotic resistance genes that make infections more difficult to treat.

Finally, the artificial constructs created by genetic engineering are designed to cross species barriers and to invade genomes, i.e., to further enhance and speed up horizontal gene transfer and recombination, the now acknowledge main route to creating new disease agents. It also means that the transgenic DNA itself may be more likely to transfer horizontally than natural DNA.

Add to that the inherent instability of transgenic DNA, which makes it more likely to break and recombine, and pieces of DNA like the CaMV 35S promoter with a ‘recombination hotspot’ that’s especially prone to breaking and recombining, and we begin to realise why we don’t need bio-terrorists when we have genetic engineers.

Despite the paucity of research dedicated to horizontal transfer of transgenic DNA, there is already evidence that transgenic DNA can be taken up by bacteria in soil and in the gut of human beings and also by gut cells. And when fed to mice, transgenic DNA can even pass through the gut and placenta, ending up in some white blood cells, cells in the spleen and liver, and in the foetus and the newborn.

The researchers stated in their paper published in 1998, (slide 14)"The consequences of foreign DNA uptake for mutagenesis [generating mutations] and oncogenesis [causing cancer] have not yet been investigated". The relevance of this remark is striking with regard to the first cancer cases identified among the recipients of gene therapy in the latter part of 2002. It makes the point that exposures to transgenic DNA carry the same risks, regardless of whether it is from gene therapy or from GM foods. Gene therapy is just the genetic modification of human beings, and uses constructs very similar to those for the genetic modification of plants and animals.

The CaMV 35S promoter was widely used in GM crops because it is a very aggressive promoter involved in replicating the virus in the cell, hence, it literally ‘forces’ the cell to over-produce the product of any gene placed downstream of it. Its recombination hotspot was discovered against a background of major doubt over the safety of viral genes incorporated into GM crops to make crops resistant to viral attack. Many of the viral genes tended to recombine with other viruses to generate new and at times super-infectious viruses.

Ewen and Pusztai had already suggested that the damage done to young rats fed GM potatoes could be due to the transformation process itself or the transgenic construct, which definitely contained the CaMV 35S promoter. We reviewed our concerns in a paper published in a scientific journal.

The promoter was widely used on the assumption that it was specific for plants and plant-like species. But it turned out to be promiscuous across Domains and kingdoms of living things, bacteria, fungi, plants and animals, humans included.

Thus, transgenic constructs with the CaMV 35S promoter might be especially prone to horizontal gene transfer and recombination, with all the attendant hazards: gene mutations due to random insertion, cancer, re-activation of dormant viruses and generation of new viruses.

Our critics say people have been eating the virus in infected cabbages and cauliflower for years without harm; and, plants are already loaded with pararetroviral sequences, not unlike CaMV, so why should an additional CaMV 35S promoter matter?

We rebutted those criticisms in a paper that was longer than the original, which appeared in the same journal soon afterwards, and no further response followed. In fact, our critics were careful never to mention the rebuttal.

We pointed out, among other things, that people have not been eating CaMV 35S promoter plucked from its natural genetic and evolutionary context and incorporated into transgenic DNA. The promoter in the viral genome does not have the same tendency to break and join up with non-homologous DNA.

The fact that plants are "loaded" with pararetroviral sequences similar to CaMV and other potentially mobile elements can only make things worse. Pararetroviruses include a family that contains the human pathogen, hepatitis B virus. The CaMV 35S promoter could activate dormant viruses like hepatitis B, which was also known to have integrated into some human genomes, and appeared to be associated with the disease.

Evidence has emerged since, that integration of foreign genes into the genome in genetic modification can indeed activate jumping genes and dormant viruses, and destabilise the genome. So we were not wide off the mark.

Behind our critics’ public denial on the safety implications of the CaMV 35S promoter, the promoter has been quietly withdrawn, and no longer appears in most GM crops under development.

Finally, what makes genetic engineering worse is that it is targeting the very mechanisms of ‘natural genetic engineering’ that coordinate the expression of genes and change the genome in non-random ways during development and in response to the ecological environment, which is what the fluid genome is about.

Imagine the genes intercommunicating and playing divine music and musical chairs in the genome. The artificial construct that has invaded the genome is like a rogue who can’t fit in, who only knows how to play a phrase of heavy metal music over and over again through the most powerful amplifier, and is capable of running amok; and all that during a rendition of life’s music that’s more akin to Mozart.

Well, the butterflies are still at it. (slide 15) That’s all, thank you.