Robert Henry Symons. 20 March 1934 −− 4 October 2006rsbm.royalsocietypublishing.org/content/roybiogmem/54/383.full.pdf · ROBERT HENRY SYMONS 20 March 1934 — 4 October 2006

October 2006 4−−Robert Henry Symons. 20 March 1934

George E. Rogers and William H. Elliott

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ROBERT HENRY SYMONS20 March 1934 — 4 October 2006

Biogr. Mems Fell. R. Soc. 54, 383–400 (2008)

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ROBERT HENRY SYMONS

20 March 1934 — 4 October 2006

Elected FRS 1988

BY GEORGE E. ROGERS AND WILLIAM H. ELLIOTT

Biochemistry Discipline, School of Molecular and Biomedical Science,

The University of Adelaide, Adelaide, South Australia, 5005

Bob Symons died in Adelaide on 4 October 2006 after a long illness. He was distinguished through his contributions to our knowledge of the structure, function and replication of plant viruses, viroids and virusoids. His research culminated in the discovery of the hammerhead folding of the RNA chain and its role as a ribozyme in self-cleavage of the RNA in some of these plant pathogens. He was a leader in his field and was responsible for commercial appli-cations of his research and the establishment in Adelaide of the first Australian company to produce and market molecular biologicals for research.

EARLY YEARS AND EDUCATION

Robert (Bob) Henry Symons was born on 20 March 1934 on the family citrus block in Merbein, northwest Victoria. Bob’s paternal great-grandfather from Somerset had emigrated from England to Australia at the time of the Gold Rush and established a family butchery business in Ballarat. Bob’s father, Henry Officer Symons, was not interested in continuing in the family butchery business and at the conclusion of his education at Ballarat Grammar School he moved to Merbein in 1921 and settled on a citrus and vines block named Dalmura. In 1927 he married Irene Olivette Wellington, who also came from Ballarat. They had three children, and Robert (in infancy known as ‘Bobbie’ later contracted to Bob) was the middle sibling; his younger and elder sisters were Helen and Judith, respectively. Bob’s early years growing up on the citrus block were happy despite the depression and World War II. He helped his father on the property and he also owned a cow that he insisted on milking every morn-ing before school. These childhood experiences ingrained in him a strong attachment to the land and were a strong influence on his subsequent involvement in agriculture and his life in agriculture-related scientific research.

doi:10.1098/rsbm.2008.0012 385 This publication is © 2008 The Royal Society



386 Biographical Memoirs

After attending Merbein Primary School, Bob won a government scholarship in 1947 to Ballarat Boys’ Grammar School and remained there as a boarder until 1951, when he matriculated. The Principal of the school at that time was Jack Dart, whom Bob respected and admired. Bob would recall those years to friends and colleagues with memories happy and not so happy, recollections that included surviving the cold winters of Ballarat. The boredom away from home at weekends was offset by roaming nearby fields and gullies with the school Principal’s dogs and rabbiting with a school friend, Ron Newland, who also came from Merbein. He recalled the boarding-house food, the quality of which was limited by postwar rationing, and he blamed his subsequent dental health on all the bread and honey he consumed for much-needed calories. He was an excellent student and participated in many sports, including being an expert rifleman. He was elected the leading scholar of his school in his final year.

Bob had an interest in studying nuclear physics but was persuaded by his father to take a course in agricultural science. He enrolled in agricultural science at the University of Melbourne in 1952 and was resident in Trinity College. The expectation was that once he had completed his degree in 1955 Bob would join his father in the family citrus business. Although he was quite at home on the Merbein property and driving a tractor, he gave much thought to his future over the ensuing year. His attraction to creativity in science led to his travelling to Melbourne to survey the opportunities for undertaking a PhD degree at Melbourne University, and in 1957 he joined Professor Frank Hird in the Biochemistry Department. His thesis was entitled ‘The metabolism of glucose and fatty acids by tissues of the sheep with particular reference to ketone body formation’. He graduated PhD in 1960.

He married Verna Lloyd in 1958, his wife of 48 years. Verna was a long-term friend and ‘the girl next door’—her family had citrus blocks on both sides of Bob’s father’s property at Merbein, and the families were good friends. Thus he began a career away from the family’s business, much as his father had moved from that of his family. From this stage in his life he became committed to fundamental and applied research on plant pathogens and a distin-guished academic career.

POSTDOCTORAL AND EARLY ACADEMIC CAREER

After completion of his PhD, Bob was awarded a Commonwealth Scientific Industrial Research Organisation (CSIRO) Postdoctoral Fellowship in 1961 to study at the Virus Research Unit in Cambridge with Roy Markham FRS, an internationally recognized plant virologist at that time, and head of the Unit. Bob’s interest in plant viruses was stimulated at that time and led to his establishing his career in that field over subsequent years in Adelaide. He applied for and was appointed to a lectureship in the Department of Agricultural Chemistry at the University of Adelaide’s Waite Agricultural Research Institute, arriving at the end of 1962. The depart-ment was headed by Professor R. K. Morton FAA, whom Bob had known during his time at the University of Melbourne, where Morton had been Reader in Biochemistry. Just at that time Morton had been offered the chair of Biochemistry on the main campus at North Terrace, and the move to the Biochemistry Department in the campus’s Darling Building occurred in early 1963. Bob moved with Morton and participated in the large task of building refurbishment, developing research programmes, funding and teaching. Progress was blighted by the death of Morton in September of that year as a result of a laboratory fire from an experiment being



Robert Henry Symons 387

conducted by Morton. Bob was working nearby in his own laboratory and rushed to the aid of Morton and his assistant. Bob was severely burned on the hands and arms from endeavouring to quell the flames surrounding Morton and it would be true to say that this humanitarian act was never really widely recognized. This tragic event was a dramatic blow to the extensive academic and infrastructural changes initiated by Morton and also to Bob’s own academic plans. The destabilizing effect of Morton’s death on the department took some time to over-come and was finally achieved after the appointment of W. H. Elliott FAA to the Biochemistry chair in 1965.

THE ADELAIDE YEARS

Family life

Bob spent the rest of his life in Adelaide after his appointment to the Waite Institute in 1962. He settled with his family in Urrbrae, having built his home only a short walk from the insti-tute, and remained residing there despite the move to the North Terrace campus. Bob and Verna raised four children, two boys (Richard and Michael) and two girls (Helen and Alison), and together they enjoyed a full and vigorous life. Verna, a science graduate of Melbourne University, was a secondary school teacher until retiring age; when the children reached adult-hood, two of them became medical doctors (Helen and Richard), the younger son (Michael) a winemaker and the younger daughter (Alison) an IT marketing manager. Bob had an early interest in wines, the growing of grapes and the virus diseases that afflict them. He maintained a large wine storage beneath the lounge room of his home; this provided much enjoyment and indeed amusement when guests visited and realized they were sitting over many stacks of fine wines. In later years Bob and Verna purchased a block of land in the Adelaide Hills and derived much pleasure from developing a vineyard that is still producing crops of Viognier and Shiraz grapes. Their closeness to the Waite Institute also led them to have a particular interest in the Arboretum at the Waite (Bob’s early agricultural background included an impressive ability to identify many eucalyptus species) and in the preservation of Urrbrae House, which is situated near the institute buildings and was once the home for the institute’s director; it is now a tourist attraction. Bob and Verna were both founding members of the Friends of Urrbrae House set up by the then director of the Waite, the late Harold Woolhouse.

The Adelaide Department of Biochemistry

From 1965, after the appointment of Bill Elliott, Bob became a major member of an academic staff that grew to eight lecturers plus postdoctoral fellows, new support staff and rapidly devel-oping honours biochemistry and PhD programmes. Increasingly, Bob’s contributions to the research and teaching strengths of the department were recognized by his elevation to Senior Lecturer in 1967, to Reader in 1973 and to a personal chair in 1987. Bob supervised some 30 PhD students, many honours students and a stream of postdoctoral fellows who formed part of his team over the years in the Department of Biochemistry and at the Waite Institute. Further, his later leadership in his field resulted in many visitors from overseas who spent study leave with him. He was a veteran in the laboratory and remained close to the bench until in his later years his research was interrupted by his ill health. His frequent presence in the laboratory had the benefit of providing additional excellence in the training of research students and earned him their respect and admiration. It was well known that because of his personal scientific standards




he reacted severely to inadequate or sloppy technique or uncritical thinking. His teaching, like his research, was centred on his major interest in the molecular biology of viruses generally, but especially on plant viruses and the much smaller infective molecules, viroids and virusoids. He was so well informed in his subject that he lectured mostly without notes, a habit that impressed undergraduates and one that early in his career he was determined to follow after observing his PhD supervisor Frank Hird, who practised the same lecturing mode.

During his academic life in Adelaide he took up several Research Fellowship awards that enabled him to undertake study leave overseas and to take his family with him. With an NIH Fellowship he spent 1971 with Professor Paul Berg (ForMemRS 1992) at Stanford University, a rewarding year in which conjointly with Berg and D. A. Jackson the joining together of DNA molecules by enzymic ligation was accomplished (13)*. This was a milestone in the manipula-tion of DNA molecules and was a basic step essential for the rapid development of molecular cloning by S. Cohen, P. Boyer and P. Berg by 1972. In 1978, Bob worked with Dr Fred Sanger FRS, Nobel laureate, at the Laboratory of Molecular Biology, Cambridge, under the auspices of a Royal Society bursary, and was involved in the early stage of Sanger’s sequencing of mitochondrial DNA.

Through the course of his scientific life Bob achieved research funding mainly from the Australian Research Council (ARC). In 1982, conjointly with three other departmental col-leagues (W. H. Elliott, G. E. Rogers and J. R. E. Wells), one of the new Commonwealth Research Centres was obtained and a Special Research Centre for Gene Technology was formed in the Biochemistry Department. His share in that new type of high-level funding by the federal government gave a considerable boost to his plant virus programmes over the next eight to nine years. It was accompanied and followed by other major funding from National Biotechnology Program Grants, 1984–88 (with the CSIRO Division of Plant Industry, Canberra, and the Institute of Medical and Veterinary Science, Adelaide), an ARC Special Centre for Basic and Applied Plant Molecular Biology, 1991–99 (with P. Langridge) and the Cooperative Research Centre for Viticulture, 1992–99.

His international distinction in plant virology was recognized by several honours and awards. He was elected a Fellow of the Australian Academy of Science in 1983, Lemberg Medallist of the Australian Biochemical Society in 1985, a Fellow of the Royal Society in 1988 and an honorary DSc of Macquarie University in 1995. His personal chair was awarded in 1987 and he was elevated to Emeritus Professor of the University of Adelaide in 2000.

Early research

Bob’s research began in animal biochemistry at Melbourne University’s Biochemistry Department with Frank Hird, his supervisor. In ruminants, microorganisms in the rumen pro-duce short-chain fatty acids, especially butyrate, from glucose and he studied the metabolism of butyrate in the gastrointestinal tract of sheep, including the mechanism of formation of ketone bodies such as acetoacetic acid (1–3). During those years he maintained his inter-est in plants and plant diseases, and that was firmly established when he spent two years in Cambridge studying plant viral ribonucleic acids with Roy Markham, one of the leaders in the field at the time. In that period, 1961–62, molecular biology was progressing rapidly, the puzzle of the triplet genetic code was being understood, and on his arrival in Adelaide Bob began his academic career furthering his commitment to plant viruses.

* Numbers in this form refer to the bibliography at the end of the text.




Although the main part of his career turned out to be directed to understanding the structure and function of viral nucleic acids in relation to infectivity and the development of plant dis-eases, he also carried out two other programmes from 1963 to 1978. One was to characterize the DNA component present in the lactate dehydrogenase–cytochrome b2 complex that had been isolated several years previously from yeast and crystallized by R. K. Morton and col-leagues. It was thought that the DNA might have some special cellular function, but in careful experiments that Bob conducted mostly with his PhD student L. A. Burgoyne (4–6) it was conclusively demonstrated that the DNA was not a specific molecule but a small degraded product from the yeast DNA, and of variable composition.

In a totally different area Bob published extensively on the mechanisms of the forma-tion of peptide bonds on bacterial and mammalian ribosomes, mainly with PhD students Ray Harris and Julian Mercer and the postdoctoral fellow Philip Greenwell in the period 1969–78 (9, 11, 12, 14–16, 19). This part of his research portfolio necessitated significant skill in designing and conducting chemical syntheses; he had already displayed those skills in the synthesis of radioactive nucleotides (see below). The work was directed to gaining knowledge of the mode of action of peptidyl transferase, the enzyme that is part of the 50S protein–RNA complexes of bacterial (and 60S mammalian) ribosomes and the binding sites for the participating transfer RNAs. Bob used the antibiotic puromycin, which was known to terminate protein synthesis by blocking the activity of peptidyl transferase. He and his group prepared several analogues of puromycin and used these to measure their level of inhibition of peptidyl transferase activity and thereby provide information about the active centre of the enzyme. It was known that the catalysis of peptide-bond formation occurred on the ribosome and involved the transfer of the nascent peptidyl-tRNA (donor substrate) in the P site to aminoacyl-tRNA (acceptor substrate) in the A site. From their findings Harris and Symons proposed a detailed model of the active centre of Escherichia coli peptidyl transferase, in which there was a binding site for the 3�-terminal CpCpA of aminoacyl-tRNA and peptidyl-tRNA, present at each of the acceptor (A�) and donor (P�) sites, respectively, of the enzyme (16).

In particular, the acceptor CpCpA-binding site was composed of sites for the terminal adenine, the first phosphoryl residue from the 3� terminus, the 3�-penultimate cytosine, and the second 3�-CMP residue. In addition, two binding sites were present on each of the A� and P� sites, one for the basic aminoacyl-R group and one for the hydrophobic aminoacyl-R group of both aminoacyl-tRNA and the carboxy-terminal amino acid residue of peptidyl-tRNA. Their improved model is shown in figure 1.

Bob was aided in this work by Philip Greenwell, a bio-organic chemist from Oxford who was interested in locating the active sites of enzymes by affinity labelling. Chemically reactive analogues of substrates or other specific ligands were synthesized and used to block the site specifically and irreversibly. Greenwell’s recruitment to Bob’s group as a Queen Elizabeth II Fellow came about because he was at Stanford University’s Biochemistry Department with George Stark at the same time as Bob was there with Paul Berg. Philip relates that they first met because Bob was foraging around the department for suitable glassware in which to conduct his nucleotide syntheses, and Stark’s laboratory was the only one then doing any synthetic organic chemistry. In Adelaide, success was achieved with a puromycin derivative in which the chemically reactive group was positioned to attach in or near the binding site for the 3�-penultimate cytosine of the aminoacyl-tRNA. Bob’s group demonstrated not only that this affinity label specifically blocked the ability of the 50S subunits to synthesize peptide




bonds but also that, once attached, the molecule was authentically in situ, its amino acid moi-ety being able to act as the acceptor in a single chain-terminating round of peptide synthesis. Furthermore, this specific labelling was found to be exclusively on the 23S rRNA rather than any of the 34 ribosomal proteins in the 50S subunit.

These findings, published in 1973 and 1974, allowed Bob’s group to conclude that the 23S rRNA has a direct role in peptidyl transferase activity and to speculate that this role might be to bind the 3� CCA terminus of the tRNA by base-pairing as in double helices. That the affin-ity label had reacted with a single 23S rRNA G residue in the presumed binding site for the tRNA 3�-penultimate C residue was confirmed in 1978 (19). At this juncture, Bob concluded that further progress in ribosome studies would require structure-elucidating techniques that were being developed by large research groups in Europe and the USA and would be far beyond the resources of his laboratory. He had, however, made a significant contribution to understanding the nature and origin of the biochemical mechanism of protein synthesis. The ultimate results of his study of peptidyl transferase were among the earliest evidence in sup-port of Francis Crick’s speculation, ‘it is tempting to wonder if the primitive ribosome could have been made entirely of RNA’ (Crick 1968). Thirty years of widespread and intensive effort have shown that this was probably true. The formation of peptide bonds between amino acids is indeed catalysed in the modern ribosome solely by 23S rRNA; in current parlance ‘the ribosome is a ribozyme’. Interestingly, Bob’s subsequent studies on plant virus RNA did much to demonstrate that certain RNA molecules can indeed exert catalytic activity.

Figure 1. Diagrammatic representation of the active centre of peptidyl transferase on the E. coli ribosome (16). The acceptor A� site is where the aminoacyl-tRNA binds with its 3� CCA terminus. The donor P� site denotes the location of the extending peptidyl-tRNA chain, also with a 3� CCA terminus. Functional groups and regions involved in the binding of the acceptor and receptor participants were deduced from studies of inhibitors and substrates and are denoted by I–IX. For example, region I is hydrophobic and attracts aromatic amino acyl groups to the A� site, whereas region II is hydrophilic and attracts tRNA loaded with basic amino acids. Similar regions were mapped for the P� site.




The preparation of 32P-labelled nucleotides

Bob’s major experimental work on plant viruses, from the very beginning (see below), included studies of RNA and DNA polymerases in viral replication. Such studies, and the later work on viroids and virusoids, demanded a constant and reliable supply of 32P-labelled ribonucleoside diphosphates and triphosphates with a high specific radioactivity (more than 1 mCi µmol�1) and also the equivalent 32P-labelled deoxynucleoside diphosphates and tri-phosphates and cyclic monophosphates. Not only were purchases from overseas suppliers in the 1960s and 1970s expensive, but the transport time to Australia also meant a loss of radioactivity. Bob developed and improved the synthesis of a range of these compounds in his laboratory over a period of 10 years and published several significant papers. His success here is a fine example of his broad range of skills from organic chemistry to viral biology. He not only supplied his personal research and that of his group with the 32P-labelled nucleotides by synthesizing the nucleotides every second week but also provided for the whole Biochemistry Department. At that time, research in molecular biology in the whole department was rapidly increasing. As noted below, nucleotide synthesis and related methods formed the basis of the establishment of the department-based university company Bresatec, which supplied these materials nationally.

Bob devised a totally chemical method that he used only when his two-step procedure was unsuitable for a particular nucleotide. The two-step method involved the chemical synthesis of nucleoside 5�-[32P]monophosphates that were then converted to the triphos-phates enzymically with the two kinases myokinase and pyruvate kinase. This method was usually more practicable, the yields of products were higher, of the order of 70–90% on the basis of the input 32P, and it had the advantage of being conducted in the same reaction flask, obviating the need to transfer highly radioactive material between vessels. In brief, for the synthesis of a labelled deoxyribonucleotide the required deoxyribonucleoside was first treated with [32P]orthophosphoric acid under condensing conditions to produce phos-phorylation of the 5�-hydroxyl group. The 5�-32P-labelled deoxyribonucleotide product was then purified by chromatography, followed by the final enzymic step to the triphosphate by addition of the diphosphate moiety with dATP as donor substrate and catalysed by a kinase. The synthesis of 32P-labelled ribonucleoside 5�-triphosphates was similar except that the 2�,3�-hydroxyl groups were protected from phosphorylation and the blocking group (O-isopropylidene) was removed after the 5� phosphorylation. The synthetic routes (17, 18) are shown in figure 2.

Plant viruses and viroids: structure, function and replication

Bob chose to use cucumber mosaic virus (CMV) as the main focus of his plant viral work. It is the type member of the genus cucumovirus group and infects several important plants besides cucumber. Tomatoes infected with it develop yellowing, mottling and curling of the leaves. During the years of these studies he used glasshouse facilities at the Waite Institute, 6 km from the Biochemistry Department but close to his home, to produce infected plants for the preparation of virus. He regularly stopped over at the Waite on his way to the main campus and taking his children to school, to check on his plants.

Bob’s studies of CMV from 1963 to 1998 ranged from sequencing of the four viral RNAs that constitute the genome, to characterization of the satellite RNAs of the virus, to characterization of viral-induced polymerases in relation to viral replication (7, 8, 10, 20–24, 30, 31, 34, 37, 44–47). Over that period he published more than 50 papers with many




students and postdoctoral fellows in international journals. A recent posthumous publication in this area was dedicated to Bob (Shi et al. 2007).

During the course of his CMV work in the mid 1970s, Bob became interested in several plant diseases, such as chrysanthemum stunt disease and avocado sunblotch disease, that were not caused by viruses (25–27). Their aetiology rests in infectious RNAs, the viroids, that infect only higher plants and are much simpler than the viruses, in that they consist of a single circular RNA molecule that can vary in length from 246 to 400 nucleotides. They are rod-shaped molecules in which the RNA strands are double-stranded through internal base-pairing but with single-strand ‘loop outs’. Unlike plant viruses, viroids are not encapsidated in a protein coat, and the RNAs are not translated into protein. The other small rod-like RNA molecules that replicate in plants and attracted Bob’s attention are the virusoids. They are also circular molecules 324–388 nucleotides long but are encapsidated in the virion of their helper virus and are known as satellite RNAs. They require their helper virus for replication and for provision of the protein coat. There are other satellite RNAs; for example, there is one associated with tobacco ringspot virus (sTRSV) that was characterized by some of Bob’s colleagues as an encapsidated circular RNA. When his research began, it was not known whether RNA-dependent RNA polymerases (RdRPs) were responsible for the replication of viroids and virusoids.

Bob was intrigued by the infectivity of RNA molecules with such simple structures and thus began an adventure into understanding their nucleotide sequences, secondary structure and replication that ultimately led to the discovery of autocatalytic RNA or self-cleavage that

Figure 2. The synthetic pathways for (a) 32P-labelled deoxyribonucleotide triphosphates and (b) 32P-labelled ribonucleoside triphosphates (17). B, base.

(a)

(b)




occurs in some of these pathogens. Bob’s publications, more than 45 papers, on viroid RNA sequences and secondary structures included studies of not only the chrysanthemum and avocado viroids but also of those that cause cadang-cadang disease in coconut palms, citrus exocortis disease and lucerne transient streak disease (28, 29, 32, 33, 35). His paper with J. Haseloff and N. Mohamed (29) on cadang-cadang disease showed that there were four RNAs, all of which are infectious. They were sequenced and their secondary structure and infectivity were consistent with their being viroids; the four were identified as homologous variants with some variation between different isolates, two being smaller (for example 246 (figure 3) and 287 nucleotides long) and the others larger (for example 492 and 574 nucleotides long). These findings were judged of such import that the RNA sequences were the front cover picture of that particular issue of Nature.

A major clue in the unfolding story of how viroids and virusoids replicate to produce more infectious particles was the finding that minus and plus RNA strands are present in infected plants. A rolling-circle mechanism for replication of the circular RNAs catalysed by an in vivo RNA-dependent RNA polymerase (RdRP) had been proposed in the literature and by the 1970s Bob had already begun studying virus-induced RdRPs. Two variations of the rolling-circle mechanism can account for the formation of plus and minus strands. In one, the plus RNA is replicated by procession of the RdRP enzyme around the plus viroid template to form concata-meric minus strands that are specifically cleaved, and the minus viroid particles are circularized by a host RNA ligase (36). The RdRP then proceeds to produce a plus concatameric strand from each minus circle, and the concatamers specifically cleave into plus unit-length fragments that are then ligated to yield the pathogenic circular progeny. The other variation was that in the first copying step of the plus circle the minus concatamer product is not cleaved but copied to a plus concatamer that is then specifically cleaved; the RNAs are then ligated to the circular progeny. Although the cleavage could be attributed to a plant ribonuclease, autocatalytic cleavage was shown to be responsible when Bob demonstrated that the RNAs from the replication of the avo-cado sunblotch viroid (ASBV) and several virusoids self-cleaved in the absence of any protein.

The virusoids studied by the Symons group included several satellite RNAs of viruses such as tobacco ring spot (sTRSV), lucerne transient streak (vLTSV), velvet tobacco mottle (vVT-MoV), Solanum nodiflorum mottle (vSNMV) and subterranean clover mottle (vSCMoV). Their RNAs were sequenced, and in 1986 Bob and colleagues (36, 38–41) showed that the secondary structures of these transcripts included a folding that in two dimensions resembled a hammerhead and contained the cleavage site (figure 4). The stability of the hammerhead structure arises from base pairing in three stems (I, II and III) and a single-stranded region in which the nucleoside cleavage site is on the 3� side, usually of a cytosine residue. The self-cleavage mechanism is a Mg2+-dependent transesterification that gives rise to fragments containing a 5�-hydroxyl and a 2�,3�-cyclic phosphate. Comparisons of sequences revealed a consensus hammerhead (figure 4c).

Figure 3. The 246-nucleotide sequence and postulated stable secondary structure of the fast variant of the four cadang-cadang viroids that causes the cadang-cadang disease of coconuts (29). The rolling-circle mechanism was proposed for the replication of this viroid.




Bob’s findings (39–41) were an extension of those of Thomas Cech (University of Colorado), who by 1982 had discovered the self-cleavage of RNA that results in the removal of an intervening sequence from ribosomal RNA in the macronuclei of the protozoan Tetrahymena. A similar self-cleavage occurs with RNA transcripts from a satellite DNA found in the newt (salamander). At about the same time, Sidney Altman (Yale University) discovered that an RNA he called RNA-P, present in E. coli, was responsible for the processing of the precursors of tRNAs, the RNAs involved in the synthesis of proteins. RNA-P brought about self-cleavage of the tRNA precursor.

These revolutionary findings established that although enzymes are usually proteins, some RNAs have catalytic activity (42, 43, 48–53). Cech and Altman were awarded the Nobel Prize in Chemistry in 1989, and catalytic RNAs were named ribozymes (see <http://nobelprize.org/nobel_prizes/chemistry/laureates/1989>).

In further studies Bob and his co-workers demonstrated, in accord with findings of Uhlenbeck (1987), that ribozyme activity could occur in trans when two separate and inde-pendent molecules combined to form a hammerhead. One example was the cleavage of a sub-strate of 41 nucleotides by a separate fragment only 13 nucleotides long in which the RNA to be cleaved is separate from the ribozyme sequence (38). This activity can occur provided that the sequence of 13 conserved nucleotides (R) and the RNA substrate (S) of known sequence, such as a transcript of a gene, can form a hammerhead structure stabilized by the three base-paired stems (figure 5).

Bob suggested that such interactions may be important in gene regulation in normal cells as well as in the genesis of symptom expression on infection by RNA pathogens through the destruction of vital cellular RNAs. The whole story of the discovery of ribozymes and the possibility that they are vestiges of the pre-protein world was a dramatic finding in molecular biology and one to which Bob made an outstanding contribution. His studies of the

Figure 4. Comparisons of the two-dimensional hammerhead structures of the plus forms of (a) encapsidated satellite virusoid RNA of lucerne transient streak virus, (b) encapsidated linear satellite RNA tobacco ringspot virus, and (c) a consensus hammerhead sequence (51). The self-cleaving sites are indicated by arrows.

(a) (b) (c)

Figure 5. An example of a hammerhead that cleaves in trans (38). The ribozyme sequence R binds to a 41-nucleotide substrate S. The arrow indicates the self-cleaving site.




hammerhead structure, and particularly its potential for activity in trans, led to the develop-ment of a potential method for cleaving RNAs in general and therefore the possibility of wide application. Jim Haseloff, a former PhD student of Bob’s, collaborated with W. L. Gerlach in CSIRO’s Division of Plant Industry and examined the self-cleavage properties of mutants of the hammerhead sequences. From the results they proposed a generic structure for in trans ribozyme activity that would cleave a separate substrate (figure 6) and demonstrated that cleavage of a specific gene transcript could be obtained both in vitro and in vivo. The struc-ture was patented by CSIRO, and the name ‘gene shears’ was coined to indicate the potential for controlling the expression of genes in vivo (Haseloff & Gerlach 1988). This ribozyme structure had an advantage over the American ones in that it was more adaptable and had the potential to destroy any known RNA sequence and especially messenger RNAs involved in the causation of diseases in plants, animals and especially humans. The hammerhead ribozyme remains a candidate for the control of gene expression in health and disease but does not seem to have the same potential as other agents such as the in vivo RNA interference mechanism of gene control that occurs in cells as described in the late 1990s (Fire et al. 1998; Hamilton & Baulcombe 1999) and for which Andrew Fire of Stanford University and Craig Mello of the University of Massachusetts, Boston, were awarded the Nobel Prize in Physiology or Medicine in 2006.

Commercialization of research and advisory roles

Bob’s development in 1966 of the methods to produce 32P-labelled ribonucleoside mono-phosphates efficiently with high specific radioactivity for his own research led to his also supplying research groups in the Biochemistry Department. Interest from around Australia in obtaining these materials there instead of from overseas became intense and led to the estab-lishment of a university-owned company, BRESA, in 1982 to supply the products regularly. The business expanded to embrace many other products for molecular biology research and in 1995 it was separated into two companies, BresaGen Ltd, which pursued basic research investigating stem-cell therapy, and BresaTec Pty Ltd, which supplied oligonucleotides and radioactive nucleotides. Bob fostered the growth of these companies. He was chairman of the board and a director of BRESA and of BresaTec in the period 1982–87 and of BresaGen to 1996. In that year the core business of synthesis and marketing of oligonucleotides as well as a range of equipment and consumables for molecular biology conducted by BresaTec Pty Ltd separated as a privately owned company, GeneWorks Pty Ltd, which continues to the present time.

In 2003–04 BresaGen altered its R&D theme and expanded its GMP fermentation facilities to the production of pharmaceutical proteins and peptides in E. coli and to process

Figure 6. The consensus hammerhead structure designed by Haseloff & Gerlach (1988) for self-cleavage for ribozyme activity in trans.




development. BresaGen ceased trading in 2005 when it became Hospira Adelaide; it continues the business of process development of quality recombinant protein and peptide products.

As described below, after his move to the Waite Institute in 1991, Bob initiated another university-owned company, Waite Diagnostics, in 1997 that continues to provide the routine diagnosis of grapevine diseases for the viticulture industry. Other commercial connections enjoyed by Bob were as a member of the Science Advisory Council of Calgene Pacific Pty Ltd and of the Scientific Advisory Council of Gene Shears Pty Ltd.

EXTERNAL SCIENTIFIC CONTRIBUTIONS

Bob filled several editorial positions over about 30 years, including Associate Editor of Virology, a member of editorial boards of Nucleic Acids Research, Plant Molecular Biology and RNA and on the advisory boards of Advances in Virus Research, The Plant Journal and Australian Journal of Grape and Wine Research.

Over the years he made significant contributions to the allocation of research funds, to research policy and to the functioning of several research organizations. He was a member of the working parties on Biotechnology and on Higher Education Research Funding of the Australian Science and Technology Council (ASTEC), in 1982 and 1986, respectively. Over the period 1986–91 he was appointed by CSIRO to act variously on the Advisory Committee, Division of Molecular Biology, on the Committee for Review of Scientific Programs, Division of Molecular Biology, and on the Advisory Committee, Division of Horticulture.

In the Australian Academy of Science he was a foundation member of the Committee on Recombinant DNA Molecules (ASCORD) in 1975. He was admitted as a Fellow of the Academy in 1983 and acted as a member of Council from 1997 to 1999.

Bob was a member, from 1982 to 1986, of the Australian Industrial Research and Development Incentives Advisory Committee (AIRDIAC, a federal government body) that was responsible for allocation of Section 39 Public Interest Grants.

He played an important role, including as chairman (1989–93), on the Biological Sciences Discipline Panel of the Australian Research Council.

At the Australian National University in the late 1980s he was a member of the Review Committee of the Research School of Biological Sciences. He also served on the Research School’s Electoral Committees for the Chairs of Molecular and Evolutionary Biology and the Chair of Molecular Biology.

Other positions he held in the 1990s included Chairman of the Board of the Australian Genome Research Facility and membership of the Council of the Australian Wine Research Institute, Urrbrae, in Adelaide.

THE LATER YEARS AT THE WAITE AGRICULTURAL RESEARCH INSTITUTE, THE WINE INDUSTRY AND GRAPE DISEASES

Bob remained in the Biochemistry Department until 1991, when at the age of 57 years he decided to move his laboratory to the Department of Plant Science at the Waite Institute. His plant virus research had reached a stage at which being within a plant science environment was an advantage, and he enjoyed a further eight years of productive research. His deep inter-




est in wines and the wine industry took him in two directions. One was to maintain the small vineyard that he ran with the help of his wife and his winemaker son, Michael. The other was to initiate routine grapevine disease diagnosis that led to the establishment of the University of Adelaide company, Waite Diagnostics, that provides a service to grape growers in the con-trol of grapevine pathogens (54, 55). On its establishment the Grape and Vine Research and Development Council provided funds for R&D, and the service receives diagnostic requests from overseas including South Africa, Germany, the USA and New Zealand. This service con-tinues to the present time under management by one of Bob’s colleagues, Dr Nuredin Habili.

The highly sensitive molecular tools for diagnosing viral and viroid plant diseases are based on designing oligonucleotides that will specifically hybridize to the RNA or DNA of a particular pathogen. Detection of hybridization requires a traceable tag, but radioactively labelled probes are not stable or safe for routine diagnostic use; this stimulated interest in non-radioactive tags. Bob first used the biotin–avidin–alkaline-phosphatase system in which the biotinylation of DNA and RNA probes was achieved by photolysis of a biotin derivative, N-(4-azido-2-nitrophenyl)-N�-(N-D-biotinyl-3-aminopropyl)-N�-methyl-1,3-propanediamine (photobiotin) onto nucleotide probes. After dot-blot hybridization with photobiotin that had one biotin molecule per 100–400 nucleotides, amounts of nucleic acid as low as 0.5 pg could be detected, a sensitivity equivalent to that achievable with 32P-labelled probes (31, 45–47). The photobiotin probes were used until they were replaced by labelling with digoxygenin. It is pertinent to note that as a result of Bob’s pioneering efforts in diagnostic technology, the avocado sunblotch viroid has been eliminated from the plant sources and seems to be extinct in Australia.

In 1994, during his time at the Waite, Bob became ill and was diagnosed with a pituitary tumour that was benign and successfully removed by surgery. He recovered well and returned to full-time work with his group. However, he developed a cerebral problem in 1996 that worsened over subsequent years and necessitated his retirement from laboratory work and finally from the Waite Institute in 2002. He was cared for at home in Urrbrae by Verna until hospitalization became inevitable. He died on 4 October 2006.

ACKNOWLEDGEMENTS

The authors are deeply grateful for the help afforded us by Verna Symons and family and for the advice of some of Bob’s past colleagues, Professor John Randles and Dr Jane Visvader, Dr Philip Greenwell, Dr Peter Palukaitis and Dr Nuredin Habili.

The frontispiece photograph is a family photograph taken in 1989 and is reproduced by courtesy of Verna Symons.

REFERENCES TO OTHER AUTHORS

Crick, F. H. C. 1968 The origin of the genetic code. J. Mol. Biol. 38, 367–379.Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. 1998 Potent and specific genetic

interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.Hamilton, A. J. & Baulcombe, D. C. 1999 A species of small antisense RNA in posttranscriptional gene silencing in

plants. Science 286, 950–952.Haseloff, J. & Gerlach, W. L. 1988 Simple RNA enzymes with new and highly specific endoribonuclease activities.

Nature 334, 585–591.




Shi, B.-J., Symons, R. H. & Paulkaitis, P. 2007 The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection. Nucleic Acids Res. 35, 1–15.

Uhlenbeck, O. C. 1987 A small catalytic oligoribonucleotide. Nature 328, 596–600.

BIBLIOGRAPHY

The following publications are those referred to directly in the text. A full bibliography is available as electronic supplementary material at http://dx.doi.org/10.1098/rsbm.2008.0012 or via http://journals.royalsociety.org.

(1) 1959 (With F. J. R. Hird) The metabolism of glucose and butyrate by the omasum of the sheep. Biochim. Biophys. Acta 35, 422–434.

(2) 1961 (With F. J. R. Hird) The mode of formation of ketone bodies from butyrate by tissue from the rumen and omasum of the sheep. Biochim. Biophys. Acta 46, 457–467.

(3) 1962 (With F. J. R. Hird) The mechanism of ketone body formation from butyrate in rat liver. Biochem. J. 84, 212–216.

(4) 1965 The DNA component of cytochrome b2. 1. Isolation of b2-DNA and the behaviour of cytochrome b2 during chromatography on DEAE-cellulose and sedimentation in a sucrose gradient. Biochim. Biophys. Acta 103, 298–310.

(5) 1966 (With B. W. Ellery) Loss of adenine during the hydrazine degradation of DNA. Nature 210, 1159–1160.

(6) (With L. A. Burgoyne) Lactate (cytochrome) dehydrogenase (crystalline, yeast). Methods Enzymol. 9, 314–321.

(7) 1968 (With J. T. May) Properties and intracellular distribution of nucleoside diphosphokinases from cucum-ber cotyledons. Phytochemistry 7, 1271–1278.

(8) (With J. M. Gilliland) Properties of a plant virus-induced RNA polymerase in cucumbers infected with cucumber mosaic virus. Virology 36, 232–240.

(9) 1969 (With R. J. Harris, L. P. Clarke, J. F. Wheldrake & W. H. Elliott) Structural requirements for inhibi-tion of polyphenylalanine synthesis by aminoacyl and nucleotidyl analogues of puromycin. Biochim. Biophys. Acta 179, 248–250.

(10) (With J. T. May & I. M. Gilliland) Plant virus-induced RNA polymerase. Properties of the enzyme partly purified from cucumber cotyledons infected with cucumber mosaic virus. Virology 39, 54–65.

(11) 1971 (With J. F. B. Mercer) The use of EEDQ (N-ethoxycarbonyl-2-ethoxy-l,2 dihydroquinoline) in the selective N4-acylation of cytidine and its derivatives. Biochim. Biophys. Acta 238, 27–30.

(12) (With R. J. Harris & J. E. Hanlon) Peptide bond formation on the ribosome. Structural requirements for inhibition of protein synthesis and of release of peptides from peptidyl-tRNA on bacterial and mam-malian ribosomes by aminoacyl and nucleotidyl analogues of puromycin. Biochim. Biophys. Acta 240, 244–262.

(13) 1972 (With D. A. Jackson & P. Berg) Biochemical method for inserting new genetic information into DNA of simian virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc. Natl Acad. Sci. USA 69, 2904–2909.

(14) (With R. J. Harris, J. F. B. Mercer & D. C. Skingle) Substrates for ribosomal peptidyl transferase: synthesis of 3�-N-aminoacyl and 5�-O-nucleotidyl analogues of puromycin. Can. J. Biochem. 50, 918–926.

(15) (With J. F. B. Mercer) Peptidyl donor substrates for ribosomal peptidyl transferase: chemical synthesis and biological activity of N-acetyl aminoacyl di- and trinucleotides. Eur. J. Biochem. 28, 38–45.

(16) 1973 (With R. J. Harris) A detailed model of the active centre of Escherichia coli peptidyl transferase. Bioorganic Chem. 2, 286–292.

(17) 1974 Synthesis of α-32P-ribo- and deoxyribonucleoside 5�-triphosphates. Methods Enzymol. 29, 102–115.(18) The synthesis of 32P-adenosine-3�,5�-cyclic phosphate and other ribo- and deoxyribonucleoside-3�,5�-

cyclic phosphates. Methods Enzymol. 38, 410–420.




(19) 1978 (With R. I. Harris, P. Greenwell, D. J. Eckermann & E. F. Vanin) The use of puromycin analogs and related compounds to probe the active center of peptidyl transferase on Escherichia coli ribosomes. Bioorg. Chem. 4, 409–436.

(20) (With T. J. Gonda) The use of hybridization analysis with complementary DNA to determine the RNA sequence homology between strains of plant viruses: its application to several strains of cucumo-viruses. Virology 88, 361–370.

(21) (With A. R. Gould) Alfalfa mosaic virus RNA. Determination of the sequence homology between the four RNA species and a comparison with the four RNA species of cucumber mosaic virus. Eur. J. Biochem. 91, 269–278.

(22) 1979 (With R. Kumarasamy) Extensive purification of the cucumber mosaic virus-induced RNA replicase. Virology 96, 622–632.

(23) (With T. J. Gonda) Cucumber mosaic virus replication in cowpea protoplasts: time course of virus, coat protein and RNA synthesis. J. Gen. Virol. 45, 723–736.

(24) 1980 (With P. Palukaitis) Nucleotide sequence homology of thirteen tobacco virus RNAs as determined by hybridization analysis with complementary DNA. Virology 107, 354–361.

(25) 1981 Avocado sunblotch viroid: primary sequence and proposed secondary structure. Nucleic Acids Res. 9, 6527–6537.

(26) 1982 (With J. E. Visvader, A. R. Gould & G. E. Bruening) Citrus exocortis viroid: sequence and secondary structure of an Australian isolate. FEBS Lett. 137, 288–292.

(27) (With J. Haseloff) Comparative sequence and structure of viroid-like RNAs of two plant viruses. Nucleic Acids Res. 10, 3681–3691.

(28) (With N. A. Mohamed, J. Haseloff & J. S. Imperial) Characterization of the different electrophoretic forms of the cadang-cadang viroid. J. Gen. Virol. 63, 181–188.

(29) (With J. Haseloff & N. A. Mohamed) Viroid RNAs of the cadang-cadang disease of coconuts. Nature 299, 316–321.

(30) 1983 (With D. S. Gill, K. H. J. Gordon & A. R. Gould) Gene content and expression of the four RNAs of cucumber mosaic virus. In Manipulation and expression of genes in eukaryotes (ed. P. Nagley, A. W. Linnane, W. J. Peacock & J. A. Pateman), pp. 373–380. Sydney: Academic Press.

(31) 1985 (With A. C. Forster, J. L. McInnes & D. C. Skingle) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucleic Acids Res. 13, 745–761.

(32) (With J. E. Visvader & A. C. Forster) Infectivity and in vitro mutagenesis of monomeric cDNA clones of citrus exocortis viroid indicates the site of processing of viroid precursors. Nucleic Acids Res. 13, 5843–5856.

(33) (With J. Haseloff, J. E. Visvader, P. Keese, P. J. Murphy, D. S. Gill, K. H. J. Gordon & G. Bruening) On the mechanism of replication of viroids, virusoids and satellite RNAs. In Subviral pathogens of plants and animals: viroids and prions (ed. K. Maramorosch & J. J. McKelvey), pp. 235–263. Orlando, FL: Academic Press.

(34) 1986 (With M. A. Rezaian) Anti-sense regions in satellite RNA of cucumber mosaic virus form stable com-plexes with the viral coat protein gene. Nucleic Acids Res. 14, 3229–3239.

(35) (With J. E. Visvader) Replication of in vitro-constructed viroid mutants: location of the pathogenicity modulating domain of citrus exocortis viroid. EMBO J. 5, 2051–2055.

(36) (With C. J. Hutchins, P. D. Rathjen & A. C. Forster) Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14, 3627–3640.

(37) 1988 (With C. Davies) Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA 3. Virology 165, 216–224.

(38) 1989 (With A. C. Jeffries) A catalytic 13-mer ribozyme. Nucleic Acids Res. 17, 1371–1377.(39) (With C. C. Sheldon) RNA stem stability in the formation of a self-cleaving hammerhead structure.

Nucleic Acids Res. 17, 5665–5677.(40) (With C. C. Sheldon) Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 17, 5679–

5685.(41) (With A. G. Rakowski) Comparative sequence studies of variants of avocado sunblotch viroid.

Virology 173, 352–356.




(42) Pathogenesis by antisense. Nature 338, 542–543.(43) Self-cleavage of RNA in the replication of small pathogens of plants and animals. Trends Biochem. Sci.

14, 445–450.(44) (With N. Habili) Evolutionary relationship between luteoviruses and other RNA plant viruses based

on sequence motifs in their putative RNA polymerases and nucleic acid helicases. Nucleic Acids Res. 17, 9543–9555.

(45) (With J. L. McInnes) Preparation and detection of non-radioactive nucleic acid and oligonucleotide probes. In Nucleic acid probes (ed. R. H. Symons), pp. 33–80. Boca Raton, FL: CRC Press.

(46) (With J. L. McInnes) Nucleic acid probes in the diagnosis of plant viruses and viroids. In Nucleic acid probes (ed. R. H. Symons), pp. 113–138. Boca Raton, FL: CRC Press.

(47) (With J. L. McInnes & N. Habili) Non-radioactive, photobiotin-labelled DNA probes for routine diag-nosis of viroids in plant extracts. J. Virol. Methods 23, 299–312.

(48) 1990 The fascination of low molecular weight pathogenic RNAs. In Seminars in virology (ed. R. H. Symons), vol. 1, pp. 75–81. San Diego, CA: Academic Press.

(49) Self-cleavage of RNA in the replication of viroids and virusoids. In Seminars in virology (ed. R. H. Symons), vol. 1, pp. 117–126. San Diego, CA: Academic Press.

(50) (With A. C. Forster, C. Davies & C. Hutchins) Characterisation of self-cleavage of viroid and virusoid RNAs. Methods Enzymol. 181, 581–607.

(51) 1992 Small catalytic RNAs. Annu. Rev. Biochem. 61, 641–671.(52) 1997 Ribozymes to the fore. [Book review.] Nature 386, 141–142.(53) Plant pathogenic RNAs and RNA catalysis. Nucleic Acids Res. 25, 2683–2689.(54) (With Y. F. Wan Chow Wah) A high sensitivity RT-PCR assay for the diagnosis of viroids in grapevines

in the field and in tissue culture. J. Virol. Methods 63, 57–69.(55) 1998 Waite Diagnostics—a service for the viticultural industry. Aust. Grapegrower Winemaker 417

(September), 44–46.



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Robert Henry Symons. 20 March 1934 −− 4 October 2006rsbm.royalsocietypublishing.org/content/roybiogmem/54/383.full.pdf · ROBERT HENRY SYMONS 20 March 1934 — 4 October 2006