Sunday, 27 July 2014

Guest Blog!

John Guest FRS
By now, those of you reading my Blog will appreciate (or at least tolerate) my attempt to play on words. In this case, I am writing about my encounter last week with Emeritus Professor John Guest FRS, former Professor in the Department of Microbiology and later, the Department of Molecular Biology and Biotechnology at the University of Sheffield. John retired from "active service" in the University several years ago (and an emeritus title is conferred, through application to the University, upon those who have brought distinction to their institution and who wish to remain professionally affiliated in some way): he, remains a frequent visitor, attending mainly when visiting academics come to deliver research seminars in Molecular Biology, or indeed in wider areas of Science. John was elected a Fellow of the Royal Society in 1986, for his work on bacterial genetics in relation to fundamental aspects of the regulation of metabolism in E.coli. I may come back to John's pioneering work on the genes and enzymes of the Krebs Cycle at a later date, but today I want to focus on his work with Charles Yanofsky, while John was a post-doctoral fellow at the University of Stanford in California. 

Studying the genes and enzymes required to synthesise the amino acid Tryptophan in E.coli, may seem a little esoteric, but this field has given us many applications in Biotechnology, and arguably the work that John published during his time in Charles Yanofsky's laboratories ranks amongst the most significant in the field of Molecular Biology. The key paper can be found at this link, where the full text is free to download and read! You wont find an extensive page on wikipedia about this work, but I believe the experiments published between 1964 and 1967 are fundamental to our understanding of the nexus of nucleotide chemistry and protein chemistry in the evolution of life. It is also important to mention the Nobel Prize winning work discussed by Rich Roberts in his guest blog, that led Roberts and Sharp to independently demonstrate that there is an interruption in this colinear relationship between nucleic acid coding information and the primary structure (amino acid sequence) in higher organisms. These interruptions give rise to exons (coding sequence) and intervening sequences (introns). In fact only 10% of our own genome appears to code for proteins or RNA molecules.

Back to some chemistry and the fact that in the 1960s it was immensely challenging to obtain the sequence of a protein: no mass spectrometry in those days: in fact John and his colleagues would have to rely on a semi manual approaches that were developed by pioneering protein chemsits including Stein and Moore in the USA and Fred Sanger in the UK, which involved complex analysis of proteolytic fragments (see RHS). Another important piece of work carried out in the laboratory of Vernon Ingram using this powerful technique of protein fingerprinting was to  establish the molecular basis of sickle cell anaemia in haemoglobin. Worse still it would be another 10 years at least before DNA sequencing would become possible (thanks in part to Fred Sanger again!), so the team had to use recombination frequencies in order to match the gene and the protein sequences. All in all, this was an experimental and intellectual tour de force and it came along as the genetic code was cracked and a few years before Masayasu Nomura reconstituted the bacterial ribosome: the site of protein synthesis. The complete set of papers published by John in while he worked in the Yonofsky lab can be found here. I recommend those of you interested in the intellectual development of modern molecular biology take a look.

John kindly emailed me an example of his original peptide fingerprinting data from his time in Yanofsky's lab. It is shown below.

I dont think you need to be an expert in protein chemistry to see that this experiment reveals a significant difference in the peptide patterns (purple spots) between the wild type protein (left) compared with the mutant (right). This is a lovely example of how a carefully planned and executed experiment can be so revealing. John has commented on the Blog below.

The story doesn't end here though. Placing this work in context is important. Previous landmark experiments had demonstrated the relationship between genes and proteins: the classic work of Beadle and Tatum on Neurospora had laid the main foundations. Crick, Brenner, Khorana (a PhD graduate of the University of Liverpool), Ochoa, Nirenberg and collaborators (see Wiki site) were all making significant progress on the determination of the Genetic Code: the relationship between the nucleotide sequences in mRNA (and the role of tRNA) in defining the sequence of amino acids in a protein chain. By the end of the 1960s, a robust model existed for the bacterial processes of replication, transcription and translation, the mechanisms underlying the central dogma were well established: DNA makes RNA makes protein, and John was a major contributor. By the end of the 1970s, there were discrepancies emerging in respect of higher organisms: Roberts and Sharp had discovered the phenomenon of splicing, in which the message (mRNA) is first processed into a mature form before the process of translation can begin. Moreover, another 10 years later, it was observed (particularly in ancient bacteria) that protein sequences are sometimes spliced! It had been discovered that some proteins lose their ends before they become fully functional, but the discovery of a mechanism in which internal sequences of protein are chopped out and the chain re-joined, was unexpected. In fact many of the DNA Polymerases used in PCR are processed in this way. The excised segments are called inteins (and the protein equivalent of exons are known as exteins). We also now know that some sequences of DNA can encode more than one protein, achieved by starting translation in a different "reading frame" or register. In order to confound us further some organisms (such as protozoa) use a normal stop codon to encode the aromatic amino acid Tryptophan (bringing us full circle to the Trp operon work in Yanofsky's lab!). 

Despite these anomalies, we still observe the main findings that John made in Charles Yanofsky's lab hold true for the vast majority of gene:protein relationships, with the caveat that splicing is generally required in higher organisms. When you carry out a BLAST search to compare your sequences, it wouldn't work if we didn't have a robust understanding of the Genetic Code and the co-linearity of the gene and protein. I hope you agree with me that the experiments of the pioneering molecular biologists after the Watson and Crick discovery represent some of the most impressive in all of Science.


  1. This comment has been removed by the author.

  2. Comment from Professor John Guest FRS.

    Never thought I’d appear in a blog! 
    It’s very staggering to remember how many man-years went into revealing things that are now taken for granted and seem so simple and obvious.  I am glad you included Vernon Ingram’s contribution and of course it was Yanofsky who had the vision and also the energy to set up the necessary methodologies.  He had the genetic expertise and he didn’t simply import protein experts from Moore and Stein’s lab but hired in turn, an enzymologist, an agricultural biochemist and a microbiologist, and we had to develop protein sequencing techniques from the literature.

    I was so very lucky because what started as a sequencing project diversified in so many directions.  The possibility that we could solve the code by studying replacements evaporated due to its degeneracy but at the time, even to prove that the in vivo codon for Gly210 is actually GGA (not GGU, GGC or GGG) and is mutated to AGA (Arg) in the A23 mutant, was a seriously important milestone.  It then provided the means for quantifying and analysing the consequences of recombination between adjacent base-pairs in a single codon (the ultimate limit).  This in turn allowed us devise a three-point cross that defined the polarity of mRNA relative to the DNA strands (difficult to explain briefly) but this had not been established at the time.
    That list of papers is a bit daunting, especially the tryptic and chymotryptic peptides, nos 2 and 3 in your list.  I remember writing them in Sheffield in what was a very grotty vermin-infested semi-basement lab, now the monoclonal lab.  They could have been much shorter but Moore and Stein set the style for JBC which insisted that compositions should be given for every peptide. The sulfhydryl peptide paper isn’t very relevant, no 4.  I had read that cyclic ethylenimine could be used to modify proteins and generate trypsin-sensitive sites at cysteines (I think). It wasn’t very useful for the sequence work but there were interesting effects on enzyme activity. 

    I did write a review of the Stanford work for a Lunteren Conference that deals with all aspects [Guest, J.R. (1967).  The relative orientation of gene, messenger RNA and polypeptide chain.  In Biochim. Biophys. Acta Library, Vol 10, Regulation of Nucleic Acid and Protein Biosynthesis, pp. 298-309.  Edited by V.V. Koningsberger and L. Bosch. Amsterdam: Elsevier Publishing Co.]  I’ll attach a my fingerprint slide for you (Top LHS) .  It shows classic chymotrypsin+trypsin digests of wild-type and trpA23 mutant tryptophan synthetase proteins.  The mutation causes a Gly210Arg substitution and you can see one peptide is displaced.  These fingerprints are on full size large sheets of Whatman paper.

    With very best wishes,  John