Accurate quantitative assessment of the benefits accruing from the genetic improvement of crops is difficult. Crop yield increases are achieved through the introduction of new, improved varieties, through improvements in cultural practices and as a consequence of interactions that occur between new varieties, cultural practices and environmental conditions. Part of the yield increases from new varieties is as a consequence of genetic plant resistance to pests and diseases. Plant breeding, which is dependent on there being available a wide relevant genetic base for each crop, has also provided considerable improvements in quality, as well as improving adaptability to a wider range of environments.

In the developed world estimated annual yield increases from the genetic improvement of crops, especially cereals, are of the order of 1–2%. Examples are given from a number of crops of hybrid varieties, of varieties with improved quality and of varieties that are genetically resistant to pests and diseases to illustrate that conventional plant breeding, allied to multi-disciplinary, strategic research, has been very effective, both economically and in protecting the environment.

Transmembrane signalling from cell surface receptors occurs by two broad mechanisms: (i) the rapid (ms) direct opening of an ion channel, where the ion channel is a component of the receptor complex (e.g. the nicotinic acetylcholine receptor); and (ii) the more slow (s) modulation of a membrane enzyme or more distant ion channel. Most of the examples of this second mechanism involve a GTP-binding protein or so–called G-protein, and the production of a second messenger. The production of nitric oxide is a special case in that it is eventually produced as a result of the activity of the second messenger ïnositol trisphosphate. The nitric oxide then diffuses into a second cell to give rise to the production of an additional ‘second’ messenger, cyclic GMP.

All of the surface receptors themselves exist as a number of subtypes. Additionally, most of the components of the second messenger systems – G-proteins, adenylyl cyclase, guanylyl cyclase, phosphoinositidase, C, inositol trisphosphate receptors, protein kinase A, protein kinase G, protein kinase C, cyclic nucleotide phosphodiesterases, and the enzymes involved in phosphatidylinositol resynthesis – occur in a number of isoforms. Furthermore, all the enzymes are controlled in their activity by a number of co-factors and other modulators. This diversity provides the potential for selective drug action, a potential which is already being exploited and which will be increasingly so in the near future.

Over 50 different neurotransmitter and hormone receptors have been cloned, transfected and characterised. Within the pharmaceutical industry, the thrust of this research is now moving from receptor cloning to focussed application of these new tools to the task of drug development. With human receptor clones now available, it is possible to target drug design to single human proteins from the inception of a drug-design project. A critical question is whether human genes will express a human pharmacology when expressed in non-human, non-neuronal cell lines. Relevant results from studies with the human serotonin 5-HT2 and 5-HT1D receptors are presented, including the cloning of a rat 5-HT1B receptor which is homologous to a human 5-HT1D receptor gene. Another issue is the relationship between agonist and antagonist binding sites. The ability to study individual human receptors in selected cell lines which are free from interfering receptors has helped to advance our understanding of this relationship. Studies comparing agonist and antagonist binding sites of the human 5-HT2 receptor are described. Finally, the fact that so many neurotransmitter receptors belong to the same gene superfamily, the G-protein-coupled or 7TM (7 transmembrane domain) receptor superfamily, allows us to form new insights about receptor structure and function. Examples where comparative studies of gene sequences are helping to predict pharmacological properties of drugs are described.

The adoption of emerging biotechnological methods in plant breeding is presented discussing specifically the relevance of cell genetics to potato improvement. The finding and use of efficient anther plant producing strains of potato (EAPP clones) is first discussed. This is followed by the presentation of recent progress toward the obtaining of diploid potato strains producing a high percentage of monohaploids from anthers. The selection of homozygous diploid regenerants (potato ‘pure lines’) by using an RFLP approach is also presented. A summary chart is presented, containing the description of an integrated breeding procedure that may be considered for future breeding of this species.

The usefulness of restriction fragment length polymorphisms (RFLPs) in plant breeding and genetics is discussed, with particular emphasis on tagging genes, map-based cloning, the assessment of genetic variability and distances, and comparative genome mapping.

The Department of Plant Breeding and Biometry has currently established tight linkages between RFLPs and more than 20 genes of economic importance. Approximately half of these genes confer resistance to major pathogens including nematodes, bacteria, fungi, and viruses. Other genes tagged are involved in various aspects of crop quality.

Locating genes with respect to DNA markers on an RFLP map provides a starting point for cloning the genes by chromosome walking. This strategy is currently being pursued for three disease-resistance genes that have been placed on the tomato RFLP map; Pto (resistance to Pseudomonas syringae), Mi (resistance to root-knot nemotodes) and Tm2a (resistance to tobacco mosaic virus). Further discussion will include the construction of a yeast artificial chromosome library and the collection of additional DNA markers in the regions of interest through RAPD analysis of nearly isogenic lines.

The assessment of genetic variability and fingerprinting varieties based on RFLP data will be briefly discussed.

Comparative genome mapping in the family Solanaceae has allowed the relationships among tomato, potato, and pepper to be unravelled. Tomato and potato share a near perfect conservation of gene order throughout their genomes. In contrast, while pepper shares most of its single copy DNA with tomato and potato, the order of these markers is highly rearranged compared with the other two species.

Guanine-nucleotide regulatory binding proteins (G-proteins) serve to transduce information from agonist-bound receptor complexes to either effector enzymes or ion-channels. Drugs that perturb the function of G-proteins may do so by one of four mechanisms, (i) They may exert negative intrinsic activity toward the G-protein. For instance, we have shown that incubation of isolated plasma-membranes with the β-adrenoceptor blocking drug sotalol blocked both GTP-stimulated and isoprenaline-stimulated adenylyl cyclase. This suggests that the empty β-adrenoceptor is capable of tonically stimulating Gsα, and therefore adenylyl cyclase; that is, empty β-adrenoceptors promote GDP-GTP exchange, (ii) They may perturb the GDP-GTP exchange reaction. For instance, certain PDE inhibitors, including SKF 94836 and rolipram, stimulate a marked increase in the pertussis toxin-catalysed NAD+-dependent ADP-ribosylation of Giα. This effect is similar to that of GDP, which promotes stabilisation of the αβγ holomer of Gi. The effect of these PDE inhibitors is completely reversed by GppNHp, which triggers afly dissociation by binding to the guanine-nucleotide binding domain of the G-protein. PDE inhibitors may serve as a class of drugs which perturb GDP-GTP exchange, (iii) They may trigger uncoupling of receptor-G-protein complexes. For instance, the polycationic drug mastoparan binds to the C-terminal end of the G-protein and mimics the effect of receptor activation by promoting GTP-γ-S binding, a reduction in pertussis toxin-catalysed ADP-ribosylation, and inhibition of adenylyl cyclase activity. Other agents, such as polyanionic drugs, bind to the receptor to promote uncoupling of receptor-mediated activation of certain G-proteins. (iv) They may alter the cross-talk mechanisms that operate between different receptor signalling systems. For instance, protein kinase C promotes the phosphorylation and inactivation of Gi. This leads to an unopposed stimulation of adenylyl cyclase via Gs and, therefore, enhanced sensitivity to agents such as glucagon. Protein kinase C inhibitors may be usefully exploited to modulate these processes which appear to be abberant in certain disease states.

Biotechnology had its initial impact on the pharmaceutical industry well before the perceived time. The use of fermentation technology to produce antibiotics was a cornerstone for the development of the industry. This event was both before cloning (BC) and before DNA (rather than after DNA – AD). Even now the antibiotic market, which is worth over 10 billion U.S. dollars a year, is the most valuable segment of the total market, (c.200 billion dollars per year). Nevertheless the impact of biotechnology in drug discovery was until recently perceived solely to be the use of recombinant DNA techniques to produce therapeutic proteins and modified versions of them by protein engineering.

There are several other places where genetic engineering is influencing drug discovery. The expression of recombinant proteins in surrogate systems (e.g. in E. coli, yeast or via baculovirus infection or in mammalian cells) provides materials for structure determination (e.g. HIV protease) and structure/function studies (e.g. various receptors). Recombinant DNA techniques are influencing assay technology by allowing access to proteins in sufficient quantity for high throughput screening.

In addition, screening organisms can be constructed where a particular protein function can be measured in a microorganism by complementation or via reporter gene expression.

Transgenic animals also illustrate the power of the technology for drug discovery. Not only will transgenic rats and mice be used as models of disease but also for efficacy and toxicological profiling. What is learned in transgenic rodents may well set the scene for somatic cell gene therapy in humans.

Pre-messenger RNA (pre-mRNA) splicing or the removal of introns from pre-mRNA transcripts is a key process in the maturation of mRNA. This process requires the assembly of a large complex of RNA and protein molecules, called the splicosome, on the pre-mRNA transcripts. Molecular and biochemical analyses of plant intron sequence and structure and of the components of the plant spliceosome are providing information on the mechanism of intron recognition and splice site selection in both monocoty-ledonous and dicotyledonous plants. This knowledge will help in gaining an understanding of phenomena such as the difference in splicing between monocotyledonous and dictoyledonous plants, the enhancement of gene expression brought about by the presence of some introns and alternative splicing. The importance of introns and pre-mRNA splicing to accurate and regulated gene expression, therefore, is of direct relevance to transgene expression and genetic manipulation.

Crop protection against pests and diseases is of prime importance and plays a major role in agricultural production both in the Developed and Developing parts of the world.

Although chemical pesticides have been in use for a long time it is only since the Second World War that a very heavy and almost exclusive reliance has been placed upon their use. This, in many cases, has resulted in the rapid build-up of resistance by insect pests to such compounds, as is illustrated by the rapidly developed resistance to the organochloride insecticides by the cotton bollworm, Heliothis virescens. Indeed, there are many examples of resistance in a major pest being observed within the first year of field use (Metcalf 1986). In some cases the indiscriminate application of pesticides has exacerbated the problem of insect herbivory where elimination of a wide range of predatory species along with the primary pests has resulted in secondary pests becoming primary pests themselves with even more devastating effects (Heinrichs & Mochida 1983).

Oncogenes can be introduced into cells which have a limited lifespan, and immortalised cell lines isolated as a result. These cell lines often retain the differentiated, tissue-specific characteristics found in the original cells and, as such, provide an important model for pharmacological studies. The techniques used to develop cell lines from rabbit kidney, mouse macrophages and rat liver are described. A preliminary characterisation of all three types of cells has been carried out, and in each case the immortalised lines described have many of the properties of the original tissue type.

Some of the most successful early applications of genetic engineering in crop improvement have been in the production of virus-resistant plants. This has been achieved not by the transfer of naturally occurring resistance genes from one plant species or variety to another but by transformation with novel resistance genes based on nucleotide sequences derived from the viruses themselves or from virus-associated nucleic acids. Transformation of plants with a DNA copy of the particle protein gene of viruses that have positive-sense single-stranded RNA genomes typically confers resistance to infection with the homologous and closely related viruses. Transformation with a gene that is transcribed to produce a benign viral satellite RNA can confer virus-specific tolerance of infection. In addition, recent work with viral poly-merase gene-related sequences offers much promise, and research is active on other strategies such as the use of virus-specific ribozymes.

Already the field trialling of plants incorporating transgenic virus resistance has begun, with encouraging results, and effects on virus spread are being studied. Deployment strategies for the resistant plants must now be devised and the conjectural hazards of growing them assessed. Genetically engineered virus resistance promises to make a major contribution to the control of plant virus diseases by non-chemical methods.

Venoms are complex mixtures of many different components, whereas a toxin is a single pure compound whose activity can be specified. Toxins often have novel, highly specific activities. Studies on toxins open the way to design therapeutically useful molecules based on the structural information obtained from the toxins.

An example of successful drug development from a component of a snake venom is that of the inhibitors of angiotensin-converting enzyme (ACE). The first ACE inhibitors were developed from work on peptides isolated from the Brazilian arrowhead viper Bothrops jaracusa that prolonged the action of bradykinin. The venom components blocked the enzyme that inactivated bradykinin; the same enzyme activated the precursor of the vasoconstrictor hormone angiotensin. Hence, a valuable new type of therapy was established by developing the leads provided by the natural toxin.

Dendrotoxins are small proteins from mamba venoms that block rapidly activating K+ channels in neurones. There are possibilities that dendrotoxin could be the basis for drug design. A small analogue that mimicked the activity of dendrotoxin in the central nervous system might boost the activity of certain neuronal pathways. Potentially, such a compound might be able to restore some of the function of systems damaged by progressive neurodegenerative diseases. Although dendrotoxin-like compounds would not be a cure for these diseases, they may provide temporary relief from symptoms.

Conversely, compounds with the specificity of dendrotoxin but with ability to activate K+ channels, rather than block them, may be a novel way of reducing abnormal electrical activity in the brain. Hence, they may form the basis of a new type of anticonvulsant drug for epilepsies.

Recent work at Strathclyde has concentrated on defining the structure-activity relationships of the naturally occurring dendrotoxins and on synthesising small analogues of the postulated ‘active site’. Preliminary results are encouraging, and it is hoped to go from green mamba venom to dendrotoxin to therapeutically useful drugs.

Ripening of tomato and other fruits involves changes in quality attributes that make them attractive to consumers. These alterations are brought about by the coordinated expression of specific genes. Ethylene, synthesised by cells of climacteric fruit at the onset of ripening, stimulates the expression of genes required for ripening to occur. Experiments with transgenic plants have shown that a 5′ flanking region from the fruit polygalacturonase (PG) gene directs the ripening-specific expression of foreign genes in tomato. Antisense genes have also been used to down-regulate expression of the PG gene, causing a reduction in pectin degradation during ripening. This reduction in PG has beneficial effects on fruit storage life and processing characteristics. Antisense technology has also been used to assign functions to previously unknown genes. This has led to the identification of the gene for ethylene forming enzyme (EFE) which catalyses the terminal step in ethylene synthesis. Detached fruit from tomato plants in which EFE is inhibited by antisense genes produce much less ethylene and ripening is greatly slowed. The rate of ripening can be restored by adding ethylene externally. These results raise the possibility of manipulating ripening of many fruits and also of controlling processes such as abscission and senescence of leaves and flowers.

The polyamine amides comprise a newly-discovered class of compounds which exhibits considerable potential for the development of selective pharmacological tools and pharmaceutical agents. In mammals and other vertebrates, they are selective, non-competitive antagonists of ionotropic glutamate receptors, but they also interact with other ionotropic receptors (e.g. nicotinic acetylcholine receptors). Thus, they are channel blockers which are selective for cation channels. We report on synthetic studies undertaken to produce hybrid analogues of these toxins based upon the argiotoxins of spider venoms and the philanthotoxins of parasitic, predatory wasp venom. The synthesis and characterisation of a mono-acylated spermine is also described. In addition, an account of current views on the many possible sites and modes of actions of the polyamine amides is presented and their potential for therapeutic neurochemistry, e.g. for the possible treatment of ischaemic damage to the nervous system, is highlighted.

The diversity of biological functions that are exerted by toxins from snake and scorpion venoms is associated with a limited number of structural frameworks. At present, one predominant basic fold has been observed among scorpion toxins whereas six folds have been found among snake toxins. Most toxin folds have the capacity to accept multiple insertions, deletions and mutations and to exert various recognition functions. We suggest that such folds may serve as guides to engineer new protein functions.

Genetic variation within a crop species is often limited and restricts improvement by conventional breeding methods. This is particularly true for environmental stresses, both biotic and abiotic. Wild relatives of crop plants, however, provide a rich source of novel variation which can be introduced into the crop. Many alien genes for biotic stress resistance have already been introduced into crops; in contrast, the genetic control of abiotic stress tolerance is poorly understood. Genetic engineering of abiotic stress tolerance in the Triticeae is the main subject discussed here with particular reference to salt tolerance in wheat and barley. Methods of alien gene transfer, including locating tolerance genes and restructuring chromosomes, are described. One of the major limitations in transferring genes for stress tolerance is the lack of good tests for resistance or tolerance which is largely due to the fact the physiological mechanisms involved are not fully understood. Genetic markers provide a new opportunity of detecting chromosome segments carrying desired genes easily and efficiently, and these will become increasingly important as the genetic maps of crop species are expanded. Although many stress genes have been located to specific chromosomes, and some have been mapped intra-chromosomally and their dominance relations determined, there is a great lack of knowledge of the control of these genes at the molecular level. Molecular studies of this type are difficult, but it is anticipated that the limitations will be overcome in the near future.

The role of computers in drug discovery depends on just how much is known about the target macromolecule. If atomic detail of the receptor is known, binding free energy differences between drug variants may be computed. Major effort is being expended in extending the area of applicability of such studies by predicting protein structure based on homologies with known protein crystal data. Where no target structure is available, computational methods can provide leads by defining transition state structures and then using the approach of molecular similarity to define stable mimics to act as blockers.

In tomato (Lycopersicon esculentum Mill.), extensive variation can be observed in genes conditioning many plant functions including disease resistance, plant development, and fruit, leaf and hypocotyl pigmentation. Genes exist that confer resistance to viruses, nematodes, bacteria and fungi. There is great value in developing a facile system for isolating such genes, and we are attempting to create a tomato gene isolation system based on insertional mutagenesis with the maize transposon Ac. Our chosen target genes (Cf-2, Cf-4, Cf-5 and Cf-9) confer resistance to specific races of the fungus Cladosporium fulvum Cooke, the causal agent of leaf mould. We have used classical and molecular techniques to map three of these genes, correcting errors in previous reports. Ac-carrying T-DNA constructs have been developed to facilitate monitoring of Ac activity in tomato, where Ac is extremely active. These constructs have been used in plant transformation experiments, and over 100 tomato lines carrying Ac have been created. More than 20 T-DNAs carrying Ac have been localised on the tomato genetic map by testing linkage to RFLP loci. This was carried out to exploit the preference of Ac for transposition to linked sites in our tagging strategy.