tag:blogger.com,1999:blog-2636689120000745345.post6968896527532380153..comments2023-08-04T10:46:43.924+01:00Comments on Liverpool Life Sciences UTC Innovation Labs: Guest Blog!Unknownnoreply@blogger.comBlogger2125tag:blogger.com,1999:blog-2636689120000745345.post-40367171338642193442014-08-04T14:37:29.338+01:002014-08-04T14:37:29.338+01:00Comment from Professor John Guest FRS.
Never thou...Comment from Professor John Guest FRS.<br /><br />Never thought I’d appear in a blog! <br />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.<br /><br />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.<br /> <br />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. <br /><br />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.<br /><br />With very best wishes, John<br />dvdhrnbyhttps://www.blogger.com/profile/15718432580363926989noreply@blogger.comtag:blogger.com,1999:blog-2636689120000745345.post-39371611726635885342014-08-03T17:06:23.868+01:002014-08-03T17:06:23.868+01:00This comment has been removed by the author.dvdhrnbyhttps://www.blogger.com/profile/15718432580363926989noreply@blogger.com