There's more than one way of reading the following article which confirms -- surprise, surprise -- that "The finest examples of powerful yet precise control of biological processes are found in living organisms, whose systems, after millions of years of evolution, are well-honed, robust, adaptable, and capable of rapid response, yet are also fail-safe, highly redundant, self-monitoring and - repairing, and subject to both automatic and executive control or veto at multiple levels."
GE by contrast... read on.
EMERGING TECHNOLOGIES: THE MINIMUM USE OF FORCE IN PLANT GENETIC ENGINEERING
ISB News Report
Kieran Elborough & Zac Hanley
The global agricultural biotech industry is based upon the constant innovations that emerge from many laboratories scattered around the world. In the early days of the biotech industry, a climate of constant experimentation and novelty led to products that have now reached farms, stores, and hospitals. Outside the context of the lab, these products have prompted a public debate unanticipated by most biotech workers. Public confidence is now the most important factor in the continuing growth of commercially-driven genetic engineering. As a nascent industry matures, so must the technology on which it draws. The next phase of technology development for this industry is to methodically improve and render routine the fundamental technologies, bringing a new precision and greater predictability to the powerful approaches available today.(1)
Comprehending The Complexity Of Natural Systems
The finest examples of powerful yet precise control of biological processes are found in living organisms, whose systems, after millions of years of evolution, are well-honed, robust, adaptable, and capable of rapid response, yet are also fail-safe, highly redundant, self-monitoring and -repairing, and subject to both automatic and executive control or veto at multiple levels. Biotechnology exploits this amazing resource by performing relatively tiny yet significant amendments. One example is the use of genes, none of which have been derived de novo from human ingenuity but are all based on a multi-million year research program carried out by the biosphere. As basic scientific knowledge grows, the manipulations possible will become more exact, the downstream effects more predictable, the products improved, and the customers reassured. When dealing with complex, intricately interacting networks, such as genomes and metabolomes, it is preferable to cooperate and coax rather than to co-opt and commandeer. By taking this approach, the next generation of agbiotech products will be able to provide more to producers and consumers, yet possess a greater `substantial equivalence' to nature's successful experiments. Plant biotechnology often requires the use of various imprecise methods of transformation to introduce additional genetic material. These processes cause severe changes to cell metabolism by disrupting existing architectures or by activating defense mechanisms designed to cope with entirely different assaults. Methods that release cells from the restraints of higher orders of hormonal control (i.e., cell culture, a prerequisite for some transformation systems) can cause wholesale and detrimental changes in metabolism via somaclonal variation, as most probably occurred in the examples most frequently cited by the anti-GM movement. Plants can also prevent the expression of virally introduced genetic material via methylation of DNA, although this can perturb the normal regulation of other genes. Such changes in the chemistry of DNA in turn activate transposons, which propagate throughout the genome with disruptive effects on all systems. This phenomenon can be exploited as a tool for functional genomics but is generally undesirable if a novel plant is to be considered substantially equivalent to an existing food crop. Undesirable outcomes also arise from the method of DNA introduction (which mimics pathogen attack) or from the random insertion of the transgene into sensitive areas of the genome, often many times per genome. In particular, the effects of imprecise insertion may not manifest themselves in early generations since different DNA error-checking mechanisms are activated during growth, reproduction, embryogenesis, and development. These outcomes impact on the time and dollar costs of any transgenic program. One strand of current research aims to reduce these effects by working within and alongside existing processes in the cell.
Finesse Not Force
A recent paper(2) demonstrates the integration of new biological knowledge with existing plant metabolic systems in a way that reduces the disruptive effect of the genetic modification process. Koprek and coworkers devised a method of hitching a ride on a natural transposon to prevent the methylation of introduced DNA. Their aim was to overcome the `silencing' observed in later generations caused by methylation of the transgene, which can occur in more than 50% of the transgenic plants in any one experiment.
To achieve this, Koprek and coworkers devised a method that locates a single copy of the transgene in the genome away from crucial genomic and metabolomic structures. The group created two lines of modified barley using conventional transformation technology: one line carried a particular transposase (the enzyme responsible for the movement of the transposon) not native to the plant, and the second line carried the transgene of interest flanked by DNA patterns that would cause the transposase to recognize and act upon the transgene. Traditional breeding between these two barley lines eventually resulted in a generation containing a large proportion of plants with single copies of the transgene relocated by the transposase to places on the chromosomes where DNA insertion was more tolerant of the genome. The rate of silencing in subsequent generations was dramatically reduced, though not eliminated. This was probably due to other, unaddressed issues such as the chemical composition of the inserted genetic information and its new location. By piggybacking on the evolutionarily-optimized transposon-mobility system, Koprek and coworkers were able to reduce the disruption experienced by the plant cell when undergoing transgenesis, and thereby achieved a considerable improvement in the efficiency of the transformation. In addition, the relocation of the inserted DNA ensured separation of the transgene and the transposase in many of the progeny, permitting easy elimination of this publicly unacceptable, second genetic element by non-transgenic breeding methods. In complementary work, Hejnar and co-workers(3) recently incorporated DNA patterns that protect the cell's own genes from being switched off by methylation (in this case, highly-methylated DNA signatures) into transgene-delivery systems and thereby prevented suppression of transgenes. As with the work of Koprek and coworkers, integration with existing systems of gene control yields an improved result, with obvious benefits to any program of plant genetic engineering.
Although the mechanism of methylation silencing activation is unelucidated, it is known to be intimately linked with an RNA surveillance mechanism termed Post Transcriptional Gene Silencing (PTGS).(4) It is also possible to work with the PTGS system in order to achieve results beyond those achieved by `brute force' approaches to gene expression manipulation. PTGS is present in most organisms, indicating a provenance in deep time, and is apparently preserved in order to prevent unwelcome production of incorrect messenger RNAs, which could disrupt the passage of correct information from genes to proteins(5) and act as a brake on viral replication.
PTGS recognizes when a given stretch of genic DNA has been transcribed in the wrong direction ('read backwards'), indicating some error in the regulation of that gene, and ensures the destruction of subsequent transcripts from that gene, whether correct or not. This system is responsible for the useful genetic modification technique called antisensing, the introduction of a new copy of an endogenous gene that is always read backwards. Antisensing results in reduced expression of the native gene but is an imprecise method of altering gene output. For some applications it is desirable to switch genes off in such a way, for example, to increase the storage time of soft fruits such as tomatoes. Smith and coworkers at CSIRO in Australia have devised a significant improvement to the antisense method that eliminates expression of the native gene completely.(6) They achieved this by integrating their antisensing into every important step of the multi-stage protein-production machinery to an unprecedented degree. Commonly, plant genes contain introns, regions of DNA that are excised after transcription by 'splicing' machinery before they are translated into proteins. The eukaryotic protein production line begins with the DNA-reading enzymes and ends with the delivery of proteins to their correct destinations within the cell, and the splicing machinery is an integral part of this system. Smith and coworkers showed that a particular antisense construction (two copies of the endogenous gene orientated towards a common center and separated by an intron) could cause the ongoing and complete destruction of all transcripts from the native gene, a level of suppression previously unattained. This 100% level of antisensing is most probably due to the full processing of the construct by the complete assembly line right up to some checkpoint guarded by the PTGS system. Constructs without the intron failed to suppress the endogenous gene completely, presumably because these constructs were not constantly associated with the processing machinery.
In support of this hypothesis, other work by Bourdon and coworkers has shown that, contrary to the textbook orthodoxy, the presence and position of introns can affect the outcome of transgenesis considerably.(7) In both cases, an improvement in effect and precision was brought about by increasing the involvement of the transgene and its products in the highly complex and interlinked information-handling and error-checking processes that have evolved in the cell. The efficacy of the genetic modification process was related to the extent that the plant's own processes were undisturbed. Acceptable Genetic Manipulation It is clear from the above discussion that the introduction of novel DNA into a genome involves the concomitant introduction of gene-derived material into other systems, processes, and mechanisms (for example, the introduction of novel protein into the proteome). All such introductions may alter the behavior of the system and, via the multi-level integration of these systems and processes, the whole cell. The latest improvements to this technology now being developed involve greater cooperation with the powerful mechanisms already set in place by evolution. By virtue of this, they afford an unprecedented level of control and precision, coupled with a sensible and desirable reduction in the disruption to the organism that is required by the industry and, most importantly, the public. This research theme will evolve further as researchers learn to work alongside other regulated intracellular operations such as chaperoning, controlled protein degradation, and cytoskeletal chromosome migration. It will not be long before, for example, the use of transgene elements such as the 35S promoter from Cauliflower Mosaic Virus to force the subjugation of cellular processes to our whim will be seen as an unnecessary and inelegant use of power, akin to the proverbial use of a sledgehammer to crack a nut.
1. Elborough KM and Hanley SZ. 2001. Emerging Technologies in Plant Biotechnology. Information Systems For Biotechnology News Report, February 2001, pp 2-4, http://www.isb.vt.edu/news/2001/news01.feb.html#feb0103.
2. Koprek T, Range S, McElroy D, Louwerse JD, Williams-Carrier RE, and Lemaux PG. 2001. Transposon-mediated single-copy gene delivery leads to increased transgene expression stability in barley. Plant Physiology 125: 1354-1362.
3. Hejnar J, HÃ¡jkovÃ¡ P, PlachÃ½ J, Elleder D, Stepanets V, and Svoboda J. 2001. CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression. Proceedings of the National Academy of Sciences (USA) 98: 565-569.
4. Di Serio F, Schöb H, Iglesias A, Tarina C, Bouldoires E, and Meins F. 2001. Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with accumulation of small RNAs. Proceedings of the National Academy of Sciences (USA) 98: 6506-6510.
5. Carthew RW. 2001. Gene Silencing by double-stranded RNA. Current Opinion in Cell Biology 13: 244-248.
6. Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG and Waterhouse PM, 2000. Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407: 319-320.7. Bourdon V, Harvey A, and Lonsdale DM. 2001. Introns and their positions affect the translational activity of mRNA in plant cells. EMBO Reports 2001 2(5): 394-398.