= A brewer’s biochemistry Part 4: Nucleic acids This is the fourth in a series of articles aiming to position malting and brewing in biochemical terms for the benefit of those who have received no training in this area of science. Readers lacking a formal scientific training will find basic chemical principles described in the first article of the series. here are two types of nuctelc acid in living ‘organisms: + deoxyribonucleic acid (ONA) ‘ribonucleic acd (ANA) In all the typos of organism that a brewer is Interested in (barley, hops, yeast, spoilage ‘organisms, themselves) the DNA comprises the Information store fr the calls. RNA constitutes the machinery than transcribe and translates that information into calularactity. ‘The essential events. surrounding these molecules are: ‘+ How isthe information passed on from one callto Its daughters when the coll divides, ‘uring growth? + How is the message in DNA translated Into cellular activity ~I.. precisely how does the ‘ode get interpreted and acted upon? For each question to be answored clearly, we need to know the basics of nucle ack’ structure, Structure of nucleic acids Nuclelo acids are polymers built up of three Sifferent types of units: ‘sugar ‘phosphoric acid bases The sugars are ribose in RNA (ergo ribonucleic acid) and deoxyribose in DNA, (deoxyribonucieic acid). Reference to article 2 inthis series wil remind you ofthe structure of ‘bose, a sugar with 5 carbons and five placesin tering structure (.. tis a pentose) Deoxyribose, as you might guess, is just ‘ribose missing one of its oxygen atoms (on carbon atom number 2. Linking carbon atom number 3 on one sugar ing to carbon atom number onthe next (goto Adenine Thymine _@4 J pom Gylosine Guanine i Adenine - Thymine ei e o) &) ‘Adenine - Ura ytosine - Guanine Nott Figure 1a: The basi structural elements of nucleic acids ‘The configuration of sugars, phosphates and bases anda schemaric representation of the Importance of base “shape” to allow fing of cytosine and guanine or of adenine with ether thymine (in DNA) or with uracil (in BNA), Figure 1b: allowable fs between bases. Figure 1e:The pairing of bases on adjacent strands of DNA and aniltusiration ofthe double helix: Inthe type of illustration shown on the let the vertical lines represent the sugar and phosphate components article 2 for the numbering rib) ie the [phosphoric aca. in this way long chains are formed, [Attached to carbon atom number 1 are the ‘bases. Those are the most complicated bite of the picture, and we won't bother with their precise chemical structure, Sufice to say that both DNA and RNA each contain four types of base. In DNA they are called adenine, thymine, cytosine and guanine. In FINA thymine is replaced by ural. ‘The oni other structural feature lam goingto mention is that RNA molecules comprise Individual, single chains whereas DNA ‘molecules consist of two of these chains intertwined inthe form ofa double helix Fig 1) (Many folkhave heard ofthe proposal ofthis by tho American James Watson and English- ‘man Francis Crick working in Cambridge, England in 1953) The two chains are linked relatively losely through an association be- ‘ween the bases on the separate strands. More specifically adenine always latches on to a ‘thymine on the opposite chain, whereas cyt sine always reaches out to a guanine. The couples fit together snugly and make for a wel- ‘ordered molecule. ‘The importance of base-base recognition ‘This selectivity of binding of the bases is the very Key to the genetic code. It allows us to answer the questions posed cari. When a cell divides this is preceded by a replication of the entire DNA in the cell Ina ‘process catalysed by specific enzymes the ‘double helix ie unwound and to each strand is linked in a new strand, leading eventual to the [production of two "daughter" strands, each Identical tothe “parent (Fig 2) co AT | ca 1 TA ‘You will appreciate that if there is, say, an ‘aderine in postion 1\on one strand, then when, tho now strand starts to be linked ta, the ‘machinery knows toattach a thymine tit. the next base is @ guanine then the machinery ‘knows what o dor“ah! cytosine” ‘And 0, in this way, the genetic code Is replicated and passed on wena cell divides. Inthe same way, the message from DNA is passed on to RNA to initiate the triggering of, Cellular functions. An enzyme unaips the DNA. ‘molecule and, using one ofthe DNA strands as. the template, an enzyme stitches togethor ‘molecules of RNA. This time, iit sees an adenine it splices @ Uracil in tothe chain, and it also pops in bose Intoad of deoxyribose. This process of converting the mastor code In DNA to the messenger (FINA) is called transcription. There are many individual messengers transcribed from a single DNA molecule, each ofthe messengers carying the entire code needed for one protein. (You will recall from article 1 that the workhorses ofthePreud AT AT AT AT AT Ib XV Zit CG cG CGCG UI il NZ fA TA TA 11 1] mT fT wo og ro ON | CG Cc-G CG cell are the proteins ~they are the enzymes, the structural units, the transportrs etc, Thus itis the proteins that are the manifestation of the code, whether thoy are the haemogiobin of a red blood cel, or the flagellum of a motile bacterium, or the hordsins of barley or the surface antigens on yeast) The sequence of NA that is transcribed Into an individual ‘messenger RNA molecules called a gene, The procass whoraby the code cariod by the ANA is shifted into a protein is called translation. The key isthe sequonce of bases (Fig), Each sequence of three bases indicates ‘specific amino aid Its called a codon. Thus, forinstance f the reading machinery (anothor ‘enzyme) sees guanine, guanine, guanine one ‘ther then it calls fer the amino acid ctualy dalivered by another transfor RNA. (There is one type of transfor RNA for each amino acid ~ clever itt) tho next three bases are uracil, cytosine, ‘and guanine then the reading machinery alerts the serine carrer to get itself tothe party, and (a) AUCGAUCGGACCCUAAGGA ... ee ved le asp arg. thr leu arg AUCGAU UGGACCCUAAGGA | tp ) ‘AUogAUSGACCCUAAGGA o TTT Ta ie asp gy pro stop Figure 4.The impact of mutagen (a) the non-mutated message (b) message obiained in messenger RNA where ‘abase change has occurred (U for C) (c)message obtained in messenger RNA where ‘abase (C) hasbeen deleted. Note that UAA is the codon that says "stop reading" ~ so every ‘codon after that point would not be read. AUCGAUCGGACCCUAAGG Vey codons Figure 2 lef). The replication of DNA ‘Anensyme starting atone end ofthe double heli tats to unzip the evo strands, and new complementary bases, together with deoxyribose and phosphate are pieced in. igure 3 (above). The bass ofthe genetic code translated into messenger RNA the enzyme splices it onto the glycine. And so (0, unt the protein molecule ls completed. ‘And so you can soe how everything hinges ‘on the precise sequence of the bases in the. DNA. Ono small change ~ just in one base ~ ‘could wreak havoc because the wrong amino acid will be “screwed!” into the protein and this can easy alter the structure ofthe protein and hence its function (as we saw in the fist article in this coi) Chromosomes ‘The DNA\n the colls that concern us (apart from bacteria - and most brewers hope not to be troubled by them) is located in the chromosomes in the cel nucleus. We've all got ‘em. Youand Ihave 46 chromosomes inmost of ‘ur cells ~in fact two complete sets of 23 ciler- lent chromosomes. It's just our reproductive calls that have ono set of 23 chromosomes —in the production of egg and sperm just one copy of each Iealaed DNA trom doner 5 + ») plasmid Prasmidoponcd onzymcaly G Pasig ot OWA sic ne “nll 3 amr G 0) ce) Transloos = = Pram) ‘gee of msrest Figure § Recombinant DNA technology ‘The yeast to be transformed has its wall removed us selected enzymes (te wall would be a barrier to DNA entry) Other enzymes are used 10 “chop” the donor DNA The gene of interest will be on one ofthese pieces. The various pieces are mixed with an opened up (“eut®) small carrier ring Of DNA called a plasmid under conditions where the ends of the individual DNA pieces and the opened ends ofthe plasmid will seal together The plasmid originates from yeast. This plasmid has certain properties that allow it to be retained by a yeast cello which itis inroduced, The wal-fee yeast is mixed with the poo! of plasmids, Those yeast ells that have received the DNA of interest (and no other) canbe selected. then regenerated. Vola: transformed yeast g September 200 + The BREWER International + wwwighorg sk yee © chromosome is packaged. When these calls ‘get together ina ertlised ogg, then one copy of teach chromosome comes from each parent, returning tothe 46-chromosome complement {ie were otherwise then every time a young'un was bor they'd havo twice as many ‘chromosomes and in all the aeons since man has been around they'd have accumulated a {airfow chromosomes by now!) ‘Saccharomyees cerevisiae has 16 chromosomes. Those strains that are used for brewing tend tobe polyploid, which means that they have multiple copies ofeach chromosome = perhaps 3 or 4. Some are aneuploid, which ‘means that they have different numbers of ‘copies ofthe various chromosomes = perhaps ot one and 4of another Genetic modification Ihave no intention of lacing this article intoany ‘areas of controversy. simply stato some facts ‘andi the reader chooses to make a judgement ‘one way or ancther, wall that's up tothem. The first thing to say is that ONA is @ reasonably consitive molocule. ts bases can react with various outside influences and bo- ‘come changed. These outside factors include Uulta-violet radiation and divorse chemical ‘compounds that are called mutagens because ‘they mutate the DNA and, as a consequence, influence its behaviour, The carcinogens actin ‘this way The mutagens might load toa loss of bases ‘orto a change in bases. For example nitrous ‘cid converts adenine to something called hypoxanthine, The latter group binds to cyto- sine and not thymine. ‘And80, when DNA replicates, the new strand will contain cytosine instead of thymine, and ‘this change will eary on to the next generation: the cytosine wil ‘how be matched by a guanine on ‘the partnering strand, 80 we now have a oytosine-guanino pair where there used to be an ‘adenine-thymine pai When that ‘mutated DNA was translated into FRNA, then the message passed ‘on would have been diferent. Lot's assume that the altered ‘base message in mossongor RNA ‘was in a triplet that should have read cytosine-guanine-guanine (Figure 4a}. That codes. for arginine. By exchanging the cytosine for uracil, the reading ‘machineryis kddedin to putting @ tryptophan residue into the protein (Figure 4b), The liketnood is that this will change tremendously the resultant protein, and likely as not it won't function. ‘The situation is even worse If the mutation leads toa deletion of cone of the base pairs (Figure 4c). “The effect Isto shift the reading frame, so that all the amino acids coded after the point where the lation occurred are probably Hos: yet rovl of wal sing into pieces. The wallisgoing to be wrong. Don't get the impression that all mutation is 2 bad thing, Utimatoly it's the reason why we're alliferont and why evolution takes the course it does. The impacts of mutation can be beneficial the altered DNA codes fora better protein = for instance, changing @ single amino ‘cid might increase the heat tolerance of an ‘enzyme = then twould be an advantage gto fan organism adapting to growth in higher temperature locations. (This typeof thing can be done in the lab ~ its called protoin engineering) Of such changes over a relative etemiy did long nooks on grates develop Mutation has been used by brewing scientists in the past to eliminate unwanted activities, @g. removal of the tendency to ‘develop hydrogen sulphide, Andi can be used profitably. in research studies into the Understanding of metabolism, We've all heard of cloning. What is it? my molecular biologi fiends (ana | do have some) will forgive me, i's essentialy a scissors {and paste job on DNA (Fig 8) Take a donor Dorganiem and arecipiont organism. Extract and chop up (using a certain typo of enzymo purchased at exorbitant price) the DNA from ndly and no ad 72> (BER INSTRUMENTS LTD the former and attach the various pieces to something called a vector. This is frequently either a virus or a bit of circular DNA that can move into and out of Cells, called a plasmid. Te plasmid-donor DNA is added tothe host organism, that has had its wall removed to make it more penetrable Inside the coll the bits of DNA {including the ‘gene we are interested in) wil thor integrate into the host chromosomes or will remain nthe vector form, provided there is a positive pressure to Keep them there, That might take the form of tagging on to the plasmid some. other factor. A popular one is the gene for copper resistance. Hf the host cli are then grown in the pros tence of a high level of copper, then only the cells containing the plasmid wil survive ~ and they will also possess the gene we are Interested in. Alternatively the host organism ‘might be one used because itis deficient in something, for example the abilty to uso a Certain amino acid because they ack a certain orayme. The plasmid or vector would then be ‘engineered so as to possess the gene for the missing enzyme and so a receiving organism ow able to use that amino acid must have taken up that gene and, whatismore, must les rapid and accurate LIVE cell count on undiluted samples n of dyes required @ NEW high resolution Model 810LC optimised for measuring BOTH fermenter samples and slurries retain it itis to continue to grow on a dit that dolberately excides the amino acid that it can't make. The cells that have successfully received the ‘gone of interest (and whichever other genes are necessary for roasons just mentioned) are purted. If they are microbes then they can be used (aw permitting) straight of. they are Cells derived rom plans then thore isthe issue ‘of regenerating the whole plant. Too complex to tackle here. ‘One thing you might have clocked though is that it must easier to jiggle with the DNA of simple organisms lke bacteria than with the likes of yeast. t's all about ploidy Ifyou try to isolate a mutant of a polyploid organism you have problems: if you knock out one gene, there is at least one other copy of the same ‘gene to fall back on. And the shortage of ‘mutants makes ita challenge to select organ isms that have received an extra gene ~ go back two paragranhs. ‘You tel me whether the targeted approaches described in this section are more or less controled than mixing completely separate pools of DNA as occurs in more traditional Approaches to developing new organisms. Note that have nat asked you to comment on hich isthe more pleasurable technique. ml The Lab BCE TS 9 Analyser 800 & 810LC Sa TMA standard for live yeast concentration TintersUcctaalclale “Telephone: +44 (0) 1970 636300.Fax:"44 (0) 1970 615455, E-Mail sles@abernstruments.couk Website: hep: wwraberinstrumentscouk The BREWER International» wnighongk «September 290