Chapter 17 Frederick Sanger
Justifiably, Sanger was the only person who was awarded two Nobel Prizes in Chemistry: one in 1958 for his work “on the structure of proteins, especially that of insulin”, and the other, in 1980, shared with Walter Gilbert and Paul Berg, for“contributions concerning the determination of base sequences in nucleic acids”.
Education
Sanger was born in 1918 in the English village of Rendcombe, where his father practiced medicine. This gave Sanger an awareness of biology that, by the time he went to university, grew into a serious commitment. In many ways, he was a natural and humane scientist, helped, no doubt, by his Quaker upbringing that instilled in him the principles of nonviolence and the value of truth. He had a solid training in biochemistry at Cambridge, but it was his time as a Ph.D. student in the Biochemistry Department with Albert Neuberger and then in Charles Chibnall’s group that his commitment to research began, and his interest in proteins and their chemistry blossomed.
Reviewing Sanger’s papers is to visit one of the early threads of modern biology. The papers, with their intensely chemical character, remind one just how critical that discipline was in bringing the molecular dimension to biology. Between 1942 and 1945, he wrote five papers on nitrogen metabolism with Neuberger, almost all published in the Biochemical Journal. The last two addressed lysine metabolism; this was good preparation for his following studies on the free amino acid groups in insulin, which also appeared in print in 1945. This was followed by analyses of steadily increasing complexity that led to the groundbreaking determination of insulin’s sequence in the early 1950s.
Insulin Research
In the 1930s and 1940s there were real uncertainties about the constitution of proteins, as their size and complexity posed formidable problems. Protein biosynthesis was a mystery, although the peptide linkage had been identified from hydrolysis products, a-amino groups could be associated with a chain terminus, and other linkages, such as those in cyclic dipeptides, had been identified. Sanger’s early protein chemistry papers, written from 1945 onwards, are characterized by rigorous analysis [crystallization and elemental analysis of the DNP (2,4-dinitrophenyl)-reacted insulin and amino acids, see that confirmed their identification by the then new chromatographic methods. The rapid advance in the scope of chemical analysis of proteins followed the developments in radioactive labelling, chromatography and the enzymatic preparation of peptide fragments. Reflecting these, the papers contain drawings of the two-dimensional chromatographic patterns formed by the various hydrolysates, simple but secure evidence for the chemical conclusions - although not all the chemists of the time thought so.
Sanger recognized the need to develop new chemical techniques, and with consistent good judgement took existing and new methods of chemical analysis and used them with unmatched efficacy. Two techniques stand out: the chromatographic techniques of Archer Martin and Richard Synge, crucial in generating purifiable fragments for protein analysis and later for nucleic acids; and the thin-layer one-dimensional gels that were essential for high throughput. Although the methods he developed in protein sequencing are now not much used, the methods he developed for nucleic acids are still the basis of modem large-scale DNA sequencing and they are employed on a prodigious scale across the world.
It is striking to see how critical the chromatographic methods were in defining the order of the amino acids, seen, for example, in the 1949 paper on insulin, published in the Biochemical Journal. The paper is a landmark. Its opening sentence reads, “One of the outstanding problems of protein chemistry is the elucidation of the relative positions occupied by the amino acid residues in the protein molecule.” The paper addresses this problem and demonstrates that the insulin terminal peptide sequences in differently derived fragments are linear and have identical amino acid organization. Thus it revealed directly, for the first time, the consistent nature of amino acid organization in a protein and allowed Sanger to state, “...the insulin molecule is built up of two pairs of very similar, if not identical, polypeptide chains.”
It is a remarkable paper; however, the determination of the amino acid sequence of the insulin A and B chains was the eye-catching achievement.
It took a further year to establish the position of the amide groups and the connectivity of the cysteines, which depended on the exacting analysis of cleaved peptides containing the cysteine bonds. These results were confused by disulphide exchange-interesting enough chemistry, but requiring careful control of conditions. The findings appear to be secure on reading the paper, but even so, when Sanger examined the insulin crystal structure after its determination in 1969 in Oxford, the first thing he did was to check the disulphides and he admitted to feeling relief when their linkage matched his own conclusions.
Sanger had hoped that the insulin sequence would give at least a clue about its biological function, but it did not. However, it is hard to overestimate the fundamental importance of the determination of the insulin sequence conceptually and practically. The research defined the nature of the polypeptide chain in protein molecules and it illustrated how the chemical complexities of the protein surface could be investigated and analyzed. One less obvious consequence of insulin’s sequence determination was the challenge it threw to chemists: four groups, one in China, one in Germany and two in the USA, were stimulated to undertake the hormone’s chemical synthesis. This was accomplished in the 1960s, ten years later.
In a general way, the ability to sequence proteins made it possible to investigate the role of amino acids in mechanism and function of proteins and to establish evolutionary relationships at the molecular level. This led to some relatively simple but powerful studies, in which radioactive labelling was combined with two-dimensional separation on the controlled acid hydrolysates of the proteases trypsin, elastase and chymotrypsin. The analysis showed that there was an identical amino acid sequence at the active site in all three enzymes, demonstrating that they had diverged from a common ancestral protease.
RNA Research
The completion of the insulin sequence left Sanger looking for new directions, and at about this time he moved to the LMB (Laboratory of Molecular Biology),Cambridge. This had a remarkable scientific environment that included such people as Francis Crick, Sydney Brenner and John Smith, who had unique expertise in nucleic acid research. It was understood that the DNA bases translated via specific triplets into amino acids, but the code was still being defined (it was completed in 1966). Nonetheless, the obvious candidate for future research was DNA and in 1960 Sanger began his second research phase: nucleic acid sequencing. He began his attack on nucleic acids by addressing RNA, because in this system there were identified and characterizable oligomers available. He started looking at defined RNA structures, such as transfer RNA and ribosomal RNA, but since he was interested in general methods of sequencing, he also investigated the use of RNA-cleaving enzymes to make partial digests. In 1965, Sanger showed that it was possible to separate radioactively labeled di-, tri- and tetra-nucleotides prepared by RNase digestion with two-dimensional ionophoretic techniques. In 1967, low-molecular mass ribosome sequences were published, and by two years later the sequence of the 5S ribosomal RNA molecule had been determined. This achievement was followed rapidly by studies on the 16S and 23S ribosomal RNA molecules.
DNA Research
The successes with RNA encouraged Sanger to tackle DNA, “the instructions for living matter,” but there were still major experimental problems to solve. The commitment by Sanger to DNA sequencing, even after his wonderful successes with RNA, was for some an unexpected and even an unwise decision; it meant leaving the still very rich research possibilities in proteins. Dorothy Hodgkin reflected this view, commenting to me that Sanger’s decision to abandon protein chemistry surprised her; she considered that proteins had such interesting chemistry, mechanisms and structures- unlike DNA. Moreover, protein chemistry needed him and such a move was a waste of his unique chemical skills. When Sanger’s research plans for undertaking the sequence determination of a bacteriophage genome were reviewed, the referees were generally uneasy about such a large program, without any precedent and demanding much new chemistry. However, I understand that one American reviewer, when consulted because of these concerns, simply replied, “Fred Sanger continues to bang his head against brick walls. And the walls keep falling down.” He was right.
In order for DNA sequencing to progress, a general method to obtain suitable fragments was needed; thus Sanger focused attention on DNA polymerase and techniques to control its synthetic and degradative actions. This was one of the critical developments in his research. It led to the establishment of the so-called “plus and minus” method. It was based on limited polymerase degradation of the radioactively labelled fragment in the presence of a single nucleotide triphosphate “plus”, and on limited polymerization with one trinucleotide absent, “minus”. In this method, the segment to be sequenced is thus enzymatically generated in segments of random length that can be separated by size.
At more or less the same time, another critical development occurred. This was the use of electrophoresis on thin acrylamide gels to separate the DNA fragments. The system could handle DNA fragments of up to 300 bases and it made possible a convenient one-dimensional method for deriving DNA sequences, one of Sanger’s great hopes and critical for coping with the huge size of DNA. And with the discovery of the restriction enzymes, the problem of preparing specific suitably sized, readily purified DNA fragments was solved. The culminating discovery in nucleic acid sequencing, however, was the use of the dideoxy nucleotides which, when incorporated into the growing DNA chain, stopped further polymerization very efficiently. This method worked beautifully in producing fragments of random lengths. The dideoxy chain termination method was cleaner; it produced longer and more uniform bands on the gels that were easier to analyze than those from the plus and minus method.
In 1977, Sanger and his colleagues reported the sequence of the bacteriophage φX174 genome, a single-stranded DNA comprising approx. 5375 bases. This was another landmark. As well as providing an immensely rich resource that gave a fundamental route into understanding the organism’s metabolism, analysis of the genome revealed the most surprising phenomenon of overlapping gene sequences. This monumental achievement was followed in 1981 by the sequencing (16569 bases) of the double-stranded DNA of the human mitochondrial genome. Again, there were fundamental surprises. First, the genetic code was not universal; in the mitochondrion it is different from that of the cell. Secondly, the mitochondrial genome proved to have an extraordinarily compact organization for which, as yet, there appears to be no explanation.
At the same time, a chemical approach to determine nucleic acid sequences was being developed in Harvard by Walter Gilbert and Alan Maxam. This method was based on partially carried out selective or preferential chemical cleavage at each of the four nucleotides. The four reactions specific to the bases were carried out separately and the products run on very thin gels (developed by Sanger’s group), making comparison and direct deduction of the sequence very convenient. The method was, and still is, widely used. Most importantly, the two methods complement each other and have provided independent confirmation of their validity.
Life and Honors
Sanger does not have flashy symbols of success such as luxury cars, expensive furniture, or landscaped gardens. A socialist and conscientious objector, Sanger even declined a knighthood, although he did not care to be called Sir:“A knighthood makes you different, doesn’t it? And I don’t want to be different. But I did accept an Order of Merit, which is higher, so I suppose there’s a bit of snobbery there.” Fred Sanger was always disinclined to talk. Contrary to many researchers who like to seek the limelight at conferences by endless lecturing and talking, Sanger was a taciturn man who worked at the lab bench with his hands. He published only a few papers, for he spent ten years to elucidate the structure of the bovine insulin molecule, and nearly seven years on the sequence of DNA. His papers were read eagerly by biochemists, because of the amazing work done. He went to work in Medical Research Council laboratory in Cambridge by an old bicycle, without stylish clothing. In the hallway of the research building, he could queue up for using an ultraviolet light box to illuminate the sample spots on a chromatography paper sheet.
As an individual, Sanger was quiet and utterly serious about his work. Dorothy Hodgkin once expressed the hope that there would always be some people who would live modest lives and do serious things-Sanger, it seems to me, embodies this tradition. He retired in 1987, and retire he did. The activity he most enjoyed was doing research at the bench, he did not like writing or teaching and was uncomfortable with administration. He claimed that he was not an intellectual, but that he preferred to do things, and certainly his writings emphasize how much he enjoyed doing experiments. Thus we were lucky that Fred Sanger, with his incomparable gifts, was able to find an environment with the MRC and LMB in which he could work so happily and effectively. The outcome was much more than the two Nobel prizes and other academic honors; it was an unequalled foundation for the most prodigious scientific explosion that is modem biology. There is no better recognition of this man than the name of the Wellcome Trust centre for genome research at Hinxton-the Sanger Institute.