Tobacco Documents Online

~.S;SlYJ~ ~YStC» G Cj~D i/a . . ~ -~~ • ~.... "~' A ..; QQ••~, ~ ~Y,st~ ~ ,l 0; ~s~xc4gu ~ L - _ _ M - JAiCt r tCa~ca ~U7inns f ~7r tt a Ly Y t»k7 k a; Ic;t . 7Sd ~ iti1+1TRJeaY tJi'fM~tifla~tid . : Ccy+n.... 1$ ~`{1KJ '';lir ')pu2~r "rTJah~~lef47t awyt - ; //~~ I~~ e ei+?T u• t;o' 1!1 .i!' ivr ~yt ~ T l Y t+ itto zc;. y'~7~a~ t vsi ,., !' ~   r* T tt 4}) r. t . t. rhYIX' a c,: , nd .~ui;~J a s.arfit r a' . ttt It~.,• qII« ~ . . . . _b : .. . . .~ ..._.., -.., . . ~a;?aF a .~':x,` :~1T,F+bc~~ t y?:.c 8J~`{a ~Ja:}ypx"C+t .:: - . : ~. _a~: CDA` Fall r Meeting ?i~ , tiTt c : ~- Ob 24 (p -ctocr-, 1977 ~ls 1 w! k . lp~+ i te•4 B ~~,Y y* hRFSTL~Q,pC{ lfzu}; Fa ~': C ~ , , ..,. c 7 pL Ta` s P' : Ec 93t~~# ~t~'ie7Twu+ . ..3a .fa~F r..cjt tP ~~E ~..~p + 0ISED ~ L o.ve w*ru J]l~a  , t.,u'K:~fJ.rv{..r7*~•ly+ilgG1 + ~~ ":r NUp1 ':1w~C,r ;Y x , ta t7 _'.. » r;• n c .: . . ~ .. . - . . ~ . ~ . - . ~.~ wtq s. {u cpey7°~E fi!rwr•t.1 1•ii.4 .:uJi+.~f•ut}'.~. 7?J C1~ Z+•'+i1"SU.K':.AL+ C The" ~Io~iriestead~~- ~' Hot SPrin8 s, V ir&inia b:; ou R- C„4s [~Q mM1 '<) . ; u J, ~, , _~ '; . t+u rJr c ~ , •r. a a • . . . . . i • r.,fk c#; : A:Y:: ry: COMMERCIAL DEVELOPMENT ASSOCIATION, INC.• 4 sZZQ(~0T 999 BEDFORD ST. . STAMFORD, CT 06905 .?fr William F. Amon, Jr: Directo_r,-_New Business Development~: Cetus Corporation -8erkeley, California 94710:' It's a real pleaeure to welcome you all to The`Homestead for what I think you will find to be a different type of meeting for the Comnercial Develop-'• aent Association.• It will be different in both subetance and format. '~'..;As to substance,'iie'11 be lookin at a7challen e and o rtunit bein'~ ~k ` Y k g g_ pPo Y 9":.`: thrust upon us as new business development people by an ever accelerating deluge of discoveries coming out of the fields of genetics and biology > areas normally outside our scope of business involvement. However, we will have ae at least one familiar base of reference: "fermentation," a techno- logy long considered the province of not only the industrial microbiologist °'. but of chemically-oriented people. We'll be looking at a technical revolu :. tion that is impacting in its broadest sense on life pzocesees and, as ; , wa're reminded in the daily press, on iife itself. ]V~d, in a narrower . sense we'll be considering the projected impact of what some call "the new _"biology" on the chemical and chemical-related industries. Specifically, we'll be looking at what's happening in biology, why it's exciting and what „ business opportunities it holds out for our respective companies. ; As to forstat, we've attempted to provide you with a program which involves a developing theme - one where to a large extent each speaker draws, builds or elaborates on the inputs of earlier speakers. To insure a good under- standing by non-biologists - by technical, business-oriented people - of what's going on in the biosciences, several of our speakers have been asked to fill an educational rather than commercial developmont,role. sasically, although each such teacher is actively involved in some specific way in cosmercializing microbiology, his main role today and tomorrow is to see - that each of us gets a"feel" for the new or developing biology and thus is better positioned to explore its potential. One result of this meeting, we hope, will be that each of you will leave Hot Springs with one tantalizing question foresost in his mind. !'What profit opportunities lie ahead for mY company as a consequence of improved biosystem capabilities7".,, . Lookinq ahead at cannsrcial'potential for`'new or developing tech_nology is. indeed, a proper role for the commercial development profession. Unfortun- ately, it's frequently a subject matter which we in CDA address in the ab- i' stract for most of our managements are reluctant..logically, to have us c; talk before the fact about what we see as promising up ahead. Thus, in this respect also, this meeting will be somewhat different. In a way, too,' it'a an experiment for your Association, so I'm sure that after the close Y. of this meeting your directors will wantl'. some , reaction,from you. - -_. . . . .; . - Now, let me try to set the stage for our speakers.. For more than a year some of you have been aware that your program chairman had changed his business affiliation to pursue a new commercial development challenge. And, I guess, I've had more of those than many in our profession. But~ as one of my good friends in the audience asked with some surprisee "Now,bu 37 what in the world is a new business development mai~ doing with a -3_ group of microbiologists?" My answer, of couree, was "Fpll, that's where it's at today*" and, in a very real ssnse, the purpose of this meeting is°' to expand on that response. . ,~ _~.5-'M- ~ t, r I must tell' yoa";Yhst I diseovered sbout' two' years 'ago.• k First'," 1' found tha0ti~ •". in oy day-to-0ay business involvements and even in my technical reading I ti.

had developed an effective mental screen against anything that ras'not , ~ specialty chemical, polymer, market development or acquisitions-oriented. For a new business development man, particularly one looking at that time'= for consulting clienta, that was bad, so consciously, I worked at eliminat- ing that screen. Specifically, I started to do more in the way of reading rather than scanning the literature. I was amazed at what I had been skipping. Most significant, I thought, was the increased frequency with which biology-related articles were appearing in C&E News, Chemtech and Chemical Week. I recall, for example, a truly superb review on enzyme - - technology which appeared (Aug. 18, 1975) in C&E News. This I clipped for my file. And, in the months following, I continued to build my file with articles on high fructose corn syrup, possible chemlcals from cellulose via cellulase, single cell protein from methane or alcohol, energy from biomass, nitrogen fixation, etc. I didn't realize, of course, that microbial pro- cesses were already responsible for 6% or 84 billion dollars of the U.S. gross national product. But it did dawn on me that microbiology was really starting to accelerate its impact on the chemical and food industries. - Then, by chance, I responded to an ad from a microbiological company, Cetus Corporation, seeking help in the chemical and food fields. Follow-up discussions were highly stimulating, for I found that little Cetus - a company I'd never heard of - was right at the forefront of what was, in fact, an exploding technology. While I'd been working in my own narrow chemical field, Nobel prizes - more than two dozen in the last twenty years - had been awarded for very exciting discoveries in the field of microbiology. Some of these were even of such nature as to affect our understanding of life and even life itself. Cetus had positioned itself to use this knowledge for profitable industrial purposes. I must ask you to forgive me if I sound as though I'm about to give a eommercial for Cetus. That's really not my intent, for, like many of you, I don't feel that this podium is the place to do that. So in telling you what I found, I'll keep it short and to the point. SRecifically, I found that Cetus was engaged in developing genetically- improved microorganisms ("bugs," as they're called in the trade) for indus- trial applications. The use of microorganisms for the benefit of mankind, of course, is not new - witness beverage alcohols, cheese, bread and vine- gar, for example. What Cetus was structured to do, however, was to engineer "bugs" in a highly efficient manner either to do better what they were already capable of doing or to do things they had never done before. 8asically, the company was built on the belief that microorganisms can do, or can be persmaded to do, almost anything the chemist can do, often much ' more, and freQncntly under more favorable conditions. And, the company was uniquely structured to pursue this belief to a profitable industrial eon- clusion. In a very real sense Cetus was in the business of inducing muta- tions and isolating desirable, super-performing microorganisms - a task which can be likened to creating "needles" and finding them in a large "haystack" environment. - Just how that's accomplished ia "the Cetus story" and'it should not be part of this introduction. Rather, your attention should be directed as vas mine to the greatly enhanced, current ability of the microbiologist - to his ability to better tailor and harness tiny living organisms for the • welfare and profit of mankind. For living cells, or the products of living cells, can and do produce bulk and specialty chemicals, antibiotics, enzymes and food products -- they convert wastes into non-pollutants or useful products - they enable us to better control disease - thoy even help us mine low grade ores - and these are all things that are of high interest to Ys. eiosystems, indeed, are part and parcel of our daily lives. And, we submit, ~. ,. On the - >; these systems are now poised for growth.' our timing seems right ~ . d,"~`~ one han~ . the micirobieloqisE'has new and improved tools"=at his disposal ' they are ready to be used bythe~chesiistland the lood technologist. On the ~ `other hand, major changes are imminent-in the world's raw materials'position. : _ = It's a bit iroic btWld nu pre-or War II, carbohydrates served as the feed - ;ostocks for the fermentative production of major antiti qu ee ot a number of ry bulk chemicalst btal id i aceone,uno,nustrial ethanol, lactic acid and glycerol, for example. Then came low cost petroleum In esaence oll .., ased chemical synthesis took Fie overermentat .on processes were rleqated '~ to the production ofill t expensve moecues or thoseoo complieated for , organic synthitibiti esaf anocs, enzymes, vitamins steroid ,s, selected f" amino acid aditiid i sn crc ac. Evenn fermentations hydrocarb on feedetocke "` were often more economic than carbohydrate alternatives. _~'I~:_ . . . . . . - . . .. . •Y Now, of couree, we are cominq about fuli oircle:`petrolaum is no'longr ,-e = ~Xh 'y e . ceap.Carbohdrates such as'starch, hydrolyzedtarch (corn syrups and'"`~''glucose) sucodl ,rse, corn an cane ewaes and oth es,er renewable resource ~} materials (eventually cellulose, perhaps lignin) are once aqaln attractive>' , YjAnd, when petroleum and petrochemicals, themselves, are used it makes more ~~ sense today that they be used for chemical or fermentation purposes rather than for heat or fuel , .i"4:"~~({& ~;^1a:cf*ey'1f,'*:Y_ ,~.,. . Thus, with new capabilities,'new technology and changed economics,'biosys= :~.tems warrant re-examination,2~ In these two days.our speakers will position d , you for this re-examination •So,,on with our program. . . . -. . u y~ p . . t. k i~. ~+`l" L~° r;! KtflsTu+ ~ a~as + s~FTC"it cr caY.,6e"~`"~'"t,f~Ctifi~~~"~} C1t+Y x~2aa+se.'~~77f .~~~t,~ }» .. aF ~P~i;+FM49 wt~ffiwkt:~eti~;~ fi~w}~f8sriaL~~~i~rx'~3pbea 9v~~µn8 3x 4 «.rrti _ r7 friii4,+ s (4;`aif¢p F+M~~F~'9p vrs+alAs?7y g~l~kZfif S s~ ~re,rl.c` {E`r hkl .cv [1.1 MA a~atvtF(^f at~ *tr!!':e }3rcRr Fa`,Javc.GduF<] a,~34l+m~sF: cv~ Vl+sxFr,iZtN c;ea+w~a+~~~ur~X p~ ~ S.~ul~as~ txu lf.rv Pkdu_;??K~7 ~ Sc sj f~ >,ck4 r f* t.'::_ t„t~;t.4> yC pa.rT:X~: ~~x$xx :`3,h wx'frtL a la:i,~ mxya~ _,.. : s+ LS4°:.} 'y~ Fyrf a T'qtlF. nS+CSr. ~P i;X9~r~icta`.,+=oi<p ~+ee~ i-a;~.~ t;:a . }vi7iq3 .._ ' ~v~:4;i^~ w ~z ci, ,_ yt Fs~;cz~r'r, aa.FFSn{~ 'r~aGn{ ,o'i#h n{) v $!r. . ,~r2x.ia~ta Q~x cn a a f:; uf ' E T~t ~'F 9 Th 'Sws4:x~t,74~ti` ~ +s:;,rv •ccv.rv~ ,~~a- ai- ., ~Fr7 ~C ~ Y 7.: y.~ +Ittf} .I y 7t . ! xol...7.n ox €y•, r.r~r(-"`; c'r+ C4 ~{~Ia-7 s~T~Faq ru rnaGc i~ s cT ~ r[ Q3~?,'. aycc . rV<,i,~T7 ~,`~uA;ryu~tb.*ta.~if ~I~~3~A.'tntr : p~,n}~a~»s+:w~ t'IJ ,<t~+a..~i~.r 9~.afL .t'~+-~F~rSiL?~t1L::jIr.~:`:.,~uLc`.~y st srs+r+scC' ;~~ ~ l~dcg~*_,r~ooc>ft 3> fE:~ tyLr~sk~t~~~a~ SI~ lfd;cs $ ~iq~~fe~, „l~Qe ~~t,3 ~~1S.rq+X4g-.~er ~?ct{Kj~t~~ i °t3;Z~~tLrc.' 1f~4 ~c fsry7tvirt~ti}c'+1G 0;4~SrxI~VF f~itt,~~~~s`t~s} VC, va ~; xrgalnt~F.~76jo a aroh~r e~=~F.~'rlui::~ Z+F~rS~~f. _ t~w:ot` rct `hT77 ~)u r:.•al ~lT~`d3..2T:ctc4'nx+f' :Fb~..~i+t~~ A6.'y.4+s~FT:Gy f..rr~ ~Ya)C~7~ Y' ~ 't iS rt F{~ErGU 1tt; 1T~ 7ret+ ~rrwi1~43Y ~t AXO~~ Yc -- ~ . ix~~ • . f. Vao~Ci ~}~f• t: .+[j r7 PVgT; ~~iW~~ jK~ ~~+ ~ F&6 t3~`c tr~+tJ r03svp~ .f : 1c ~ i-.ti1 Lt'i.C1tp'g '+sEt.l,Jfrf ?,bL r~ r'lxuFjun ~,GX~i7s~.t~k:2~1 T1~R!c~!~.. v~~ ItoaYFVG_4 ilfr:ior KWx24)#'# . 7: - . i ??.: . .. r. ~. ~ ! x a

. .~a .EF;•a:a~!-fi+~la ",la++~~. s~i ,~+ -st ; ...:, r rrlr.2 sux :4q'! H2O6YSTEMS " .a f r :~ r 7 - . ~'?~ N.O by ' r»cy .ex..rk~ F7~k<F]~n1i` ',ti v r~4~s; Donald 11' ~''•K ' c~.jr ra.+"Glaser. dw ~s v; Tw ~!~ofeeeoi of Phyaici' and Molecular Biology ~ru ~~}gAp~ ;~i Univeraity of California ., pWI-w TO ~s~ri!p: y C x:.a4tt r c, ~ s•,. Berkeley, CA 94720 i+,v.Re1•+•,;F ly,yt ~~ ~ I'm pleaaed but a little bit intimidated'to see how many poets and artists Bill Amon succeeded in conning into gdtting up early to come to - this meeting. in the past I've given a course at Berkeley which had the nickname, "Molecular Biology for Poets and Artists." That course consisted of thirty lectures, with two midterms and a final exam with the grade based mostly on the examinations. My assignment today is to summarize those thirty lectures in about a half hour, telling you what's going on in the " new biology, and then go on to describe what impact it has on the real ...,,~; world. I can't promise you that those of you who've had no contact at all „ with chemistry are going to follow everything, but, I think by the end of .: my talk you'll at least be in a position to ask interesting questions. ; I'll try to give you an impressionistic view of what's happened. 4:jt.i:Y What's called the new biology in the real world ie called molecular- biology at the university, and is a sort of mixture of biochemistry and genetice.. In fact, one distinguished biochemist calls molecular biology "the practise of biochemistry without a license." There is a certain ,~. amount of competitive feeling about this between the departments that are called Molecular Biology and those that are called eiochemisty, and_ they..: very often do similar things in universities. The whole development that we're going to talk about for the next eouple of days had its beginning philosophically with the question, "What`.• - is life?" Now, that's not a new question. It's a question people have been interested in since the beginning of serious thought. What's new now '_ is that people are asking the question in the following termst "Can we, by laboratory experiment, find out whether we can explain everything that _ living things do using only the methods of physics and chemistry?", If the ~ answer is yes, then biology is a branch of physical science. if the answer ie no then we hope to discover some sort of new and vital principle, , something novel, something, presumably, extremely shocking, which will distinquish life lrom non-life. That is really the philosophical motiva- tion behind the new biology- ~• 1 This leads to the next questiont. "How do you begin doing experiments aimed at answering a grandiose question like that?" The answer of those scientists who attacked that problem was to pick something that looked simple and try to run it into the ground. That is, they have tried to take apart completely some living thing in such a way that they come to under- atand atl the pazts, and thus can duplicate, in the laboratory, all the ' chemical and physical events that happen in that living thing. ' They then`-; claim that combining all those processes gives them everything that ex- plains the organism. The organism they chose is the bacterium, Eseherichia coli. A cartoon of the organism is shown in Figure 1. It is a thousandth of a millimeter lonq, aa shown in the diagram, which means that it is about the lower limit of what you can sae in a light microscope. With the aid of such a microscope, all you see is a eort'of capsule-like thing. This r during the course of about a half hour and • organism gets longer and longe_ then it divides in the middle by pinching down and splitting into two r• identical organisms. It can keep doing that every half hour forever, ; making an immortal line of cslls. Almost none of them die. ~.,. Fifty years ago there were seriou doubts whether this organism poe- : ~Asessed heredity as a property. it was imagined to be a bit of sel!-repro-c, ducinq matter. The organisms were very "slippery," ao that when an attempty 1 'to' r'was mde'chetaaterizsthes,'Tselentists could not pin dnyEltastt c i .ow an, •cen ( traits. ''Sometimes they were killed by penicillin, sometimes notj sometimes t you had,to feed them Vitamin B and sometimes not.. As a result, scientists• believed that these were not serious living things and that tliere was no point in studying them.r.Now, in the revolution of the last'thirty yearsj ~.' ?;:we have come to underatdnd a lot about this orqanism, and most aignifi- , ~ eantly, ~+e have found that everything E, coli does, we do too. The mechan- iam it uses is chemical.. The detailed proceases that it uses to multiply r""didll i an-survve,an evove are amostdentical to the processes in our own cells. The chromosome of E. coliAshown as a circle to the right in a , Figure"•1) is a single molecule which is chemically identical with our ownr chromosomes,. The punchline is going to be, as many of you know, that we can put pieces of 'our own chromosome 'in there and the bacterium will start ` doing human-like things. -:I'1l tell you-how that comes about. - .' " Th' thi youhldt eres oneng sou note about Figure 1,.and tha is•that the4- circular chromosome is one millimeter long..`That, of course, is impossibie,' because it's shown as being•:inside a cell which is a thousandth of a milli- "',meter long:''In fact,*the picture ian't quite honest.- The chromosome is actually a tsngled jumble of DNA.'•' To get some idea of scale, if this . tbacterium were about the size of a bathtub, that molecule of DNA would be!0 like about a mile of spaghetti sitting in the bathtub. This chromosome is-+~ ` an enormous molecule:i As children, in chemistry we always learned that~- >molecules were a few-angstoms in size - water, hydrogen, sodium, all of~;_,,p:. them. Here's one that's ten million angstroms. It really deserves the '-? { name,="macromolecule," and it is really very, very much larger than the.~~ -kinds'of molecules that one is used to in ordinary inorganic chemisty. -. " Thill i ittdarkb s moecuesmporan an remale, because it already pos-<~ ,+.:•_ sessee the first property of living things, namely it can reproduce itself.; That's one of the things that distinguishes living things from non-living things. Of course, crystals can also reproduce themselves in a certain ,- ' sense, so it's not the whole storyt but it's one property.- Let me show you ' how that happens (Figure 2).,:Everybody knowe that DNA is a double helix. That means that if you untwist the molecule, it has two linear polymera.(as - shown on the left of Figure 2), which are very boring, uninteresting, chains of phosphate and sugar. :it isn't boring, however, when you can think of that thing as a sort of a zipper, with four different kinds of hooks, all of different sizes - A, C, T, and G are their shorthand names,jLyA standing for adenine, thymine,,'cytoaine,- and guanine. .They're rather simple chemicals.::.They're sort of six sided figures - very simple ringst Y~7 some of them have:two rings that join together. But they're not compli-•4;.e; ? cated. aThey're flat,:and they fasten together in such a way that if-, /4tM ; there's'an A on one side,`there has to be a T on the other side.,,They are,a particular. rThe distance-between one pair and the next one is 3.4 ang-. ; stroms. When these chemicals are all properly paized, they zip together to make a pair of helixes.. There are ten bases for one turn in the helix. r, ryou can tell by the detail that we really know how thia molecule is put F. = together,..In fact, we know where every hydrogen atom is in that molecule,,- and that it's molecular weight ie 2.8 billion. It's a well-known, very ~ complicated thinq. Every part•of it is identical with every other part..~: Every part is equally strong.z There are no weak bonds in that moleculel they are all uniform.. There is no place a chemist would obviously go to unhook it if he were.trying to manipulate it. There are places, as you'll , see later.on, but,at this stage, they're not obvious. The DNA molecule can have any sequence of those four letters. :The`` -' sequence,carries information and is, in fact, the blueprint which makes ita possible'for the molecule to reproduce itself perfectly. Nearly everythini we ara, everything about our shape, everything about our.ehemieLry is carried.in a sequence of letters like that in our own chromosames.: TheE' molecule has a very heavy responsibility because it has to carry all that information, to reproduces itself, and it hae to last, without change, to t' ' keep the organism goinq,!or the allotted period of time. And it does ~~ ,-' Ti . a reproduction of the molecule is already contained in its struc'. ; that;,-,V 4 ;t . . ._ .. . m. „_. . , '`4. 1

~Y '7r.N!o, k ,0~~.t:•- ~~~t:v~:rial~frt~~' ,~"~. . . .% TQC'R.•IY..O•-~ - •ri~ - ~ ~~.,~,, ture because an enzyme in the pst just antered`tlie sqlacule,:opened ap.the=i zipper one hook at a time until it was stretched out and then'other enzymes which contain free floating hooks attach them on in such a way that you always get the correct hook at the right place. 'It's a very complicated _ thing when you look at it in detail. One of my-students recently showed that in this bacterium at least eleven different enzymes are involved in that process. But it's not at all understood, so sometimes when I show this Figure, I present it by placing a fig leaf over this unzipping area. That's where the action is and while we know the complexity of it, we don't •, know the details at all. It's all very well and good to call this thing a blueprint; but how in ' fact does all this information that I claim ie contained in a nequence of letters (in fact, it's only a four-letter alphabet) -- how does that do anything? The answer is that there's a chemical process (Figure 3) which copies those sequences into a chemical called RNA. RNA is almost the same as DNA - it also has four letters. The process is almost like making a tape recording copy of a primary tape. A little piece of RNA called mes-'''.. enger RNA, finds its way inside the cell and there it runs into little gadgets called ribosomes. Theee ribosomea, essentially behaving as trans- lating machines, read the'sequence of letters which the messenger RNA copied from the original DNA chromosome. The ribosome translates this; ' I coded message into new sequpncce of amino acids. Thus, these ribosomes essentially do what a machine would do that, for instance, reads a magnetic tape and prints out a punched paper tape. The magnetic tape has two let- ters; the punched paper tape has, typically, eight levels which corresponds'-- to a lot of letters. But here we're going from a four letter,alphabet, A,. T, C, G, to a twenty letter alphabet, the twenty amino acids which can be coded by various combinations of A, T, C, and G. Although it's very co_m-:' plicated, the cell contains a dictionary which translates one alphabet to': the other. If you feed this cell streptomycin, it will kill it, and the -f mechanism of killing is unbrlievable. It messes up the dictionary so that the translation from the four letter alphabet to twenty will be done incor- rectly. Thus, what originally made sense is turned into jibberiqh. The notion that a chemical can kill a living thing by interfering with the information content is really kind of a wild idea. Of course, it'a very r familiar to us in the computer business, but to find it in biology Is really surprising. " Thtkbl is that after these polypeptides arefonned te nex remerae thing, y they break away fzc.e the ribosomea and drift off by themselves. Then they fold up into sort of a three dimensional pretzel, as shown in Figure 3, with two or three turns which fill space. That pretzel is a protein and it can do many things. In a bacterium it's usually an enzyme which catalyzes a chemical reaction, but it can also be muscle, hemoglobin, insulin, hair, u~ fingernail, etc. A tremendous fraction of the important parts of ol.tr , i- bodies, and of the bacterium, are these proteins. They can take a great ~; v • variety of shapes. The chromosome of E. coli which I described has'spsethinq like four t~ w. million zipper hooks, these pairs of bases of the A, T, C, G,variety. ; ~' They're divided ur into groupq of about a thousand, or a little smaller •' than that (Figure 4), so that t_here ia enough space around lh- ~r.•laculo for " something on the order of five thousand of those groups. Each giouping of a thousand or so base pairs is a genet and each gene is read off by the ~ ~~. messenger RNA, translated, and produces one protein. Hence, we have the.. general law in biology - one gene/one protein. That's how information is organized. Figure 4 also shows what could well specify an enzyme that chews up penicillin, so that if you threaten this bug with penicillin, it starts making an enzyme which digests the penicillin and destroys it.~ That's how a bug is resistant to penicillin. Figure 4 also shows a more complicated process in which the organism senses whether there's any sugar available that can support growth. If you put one of these bugs in a beaker of warm water and add a pinch of salt and a pinch of sugar, then !t,', will st4gb%,dividing every half hour doing all these things I've mentioned. ; 6. ~Y'l . y..4 L~k~~iu~ ~ `; O._ twv.rtda ., yc g s!! has to.live onr so it has a eenaor ~,ito detect rhether.there's lactose or maltose or dextrose or;alcohol present. ~ kSeveral hundred different carbon compounds can serve adequately to_support ti;'~the cell's growth, but the cell has to know which one it's getting in order A:r.to digest it. You will also notice on Figure 4 the enzyme lactase which Idigests lactose, and the pump which pumps sugar into the cell. We know a s,lot about how theae work, but we don't know really what the molecule looks like. Suppose that the cell is offered some lactose (shown in Figure 5 as e a square) and some maltose (shown as a triangle) and one of these sugar j.molecules or several of them diffuse into the rather porous wall of the - ;ce1l. If the square one does, it can fit into the slot in that control p k thing.-- That control thing is a protein which haa just the right shape, so ::that it sticks on the DNA and keeps that portion of the DNA covered, sort ' V of locked up, so that none of that messenger RNA can be made as long as ^- that locking protein is sitting there. But when the sugar molecule somes "' ~-in, it fits into the.lock, distorts the molecule and the lock €alls off. >=When that happens, a piece of DNA is exposed, so that the cell can etart t f peeling off copies of that information. The genetic information, in this " case actually encoding three different products, is read at once.. One of - -~:those products, lactase, is an enzyme which allows the cell to use lactose Y 'as a source of energy.,,Another product, the "lactose pump,"* permits the 'O`cell to uselthe lactoae•in the environment more efficiently.- So one class n of protein diffuses and-sticks itself in the wall and starts pumping in all the lactose it can find;. Notice that the lactose rather than maltose is . P'there because of that lock. I've shown it as a conveyor beltt you can draw, it as you like. No one knows really how it works. But what it does is ~eoncentrate lactos_e..+It will ignore the trianglesp because they won't fit "4nto the machine. It's very selective. ° . ~ Once the sugar has,been pumped inside the cell,•.it:finds an'enzymev:; 'shown in Figure 5 as this thing with ugly looking jaw)iti f it s wangor. 'The enzyme breaks down the lactose molecule into galactose and glucose..:+ `" ':-Then a lot of things begin to happen. This !s typical of a control mech- ~.'. . .. . ?~anism. Another big generalization we have learned about living things;-or at least about E, eoli, !e that every process is controlledt every process has ` some kind of control element. Really sound engineering principles have ° been built into these bugs. Nothing is an open loop. There's nothing that ' is turned on and simply allowed to go. Always the quality and quantity of the process is monitored, and that information feeds back someplace in the _ _ process to change the rate or change the quality of what's being produced. "And, in fact, the differences between one organism and another reside more T''in the characteristics _ and the quality of its control process than in the . ~creal chemistry itself The chemistry of our cells of human cells is .,, almost identical with that of E. coli. The DNA is exactly the samet the f'"enzymes used are almost the same. Even though we adults don't have as much r'lactase as this cell,.newborn human infants have lots of that kind of • lactase, When they get past the nursing stage they lose that lactase more `=~or less," so that adults do not tolerate milk sugar as well as children do. That's true of all mammals, but particularly of humans. • '.' A'friend of mine in the biochemistry department at Berkeley, Alan E Wileoei, has been studying evolution for a long time, trying to compare the . 'r-'bioehemietry of salmon with that of chiekens, with that of yeasts, with tfL''that of mammals, and so on.;,His object is to see who is related to whom, rFif you set as your criterion biochemical similarities, and then see how <'-:*that agrees with the fossil records and the classical ways 9f viewing R; evolutlon..~He has established an evolutionary tree using bior,hemistry _" alone.- Itti very similar to what the paleontologists have bedn claiming ~" all along;tbut there is one shocker.- He couldn't tell the difference 1 between humans and chimps. 4The biochemistry of our insulin and their;•,_ insulin;-of our hemoglobin and their hemoglobin, of our muscle and their ``muscle,a^is all the same. His conclusion was fascinatinge namely, that the difference between chimpanzees and humans !s not in the building blocks,

~ ~ = ~.. r: • o.. tl , . ,J. ~. ~ :• " • , , .y t ., not in the composition of the biochemicalsir,but rather, the difference is in the software, that is, in the control mechanism by which these chemicals are turned on and off during the developmental process of the fertilized " - - egg to the fetus to the adult. Because of these discoveries, the focus in'- molecular biology is shifting very rapidly. Most of the people who are working on the fundamental question have lost interest in the details of biochemistry and are focussing on details of the control mechanisms. Those seem to be the things which make the finer distinctions that we're inter-d eated in. I don't think you're ready to hear about sex quite yet, so I want to'° " say one more thing. The information contained on the chromosome controls and specifies the shape of each protein molecule. If that chromosome gets hit by a cosmic ray, or by an ionizing particle from radioactive material, or by some aggressive chemical, it can change one of those bases. The i change either kills the system because the organism can't copy its DNA anydc:. more, or it simply makes a mistake. Making a mistake means that the messen=. ger RNA copies the mistake faithfully, and then the protein has a mistake in itl and that means when it folds up, it doesn't fold exactly the right iz way. It's very important to remember that the exact three-dimensional "' structure you get for protein is determined completely by the sequence of ~" amino acids. If you change that sequence, you get a new protein. Most of > the time, mistakes are lethal - the protein doesn't do what it's supposed `; to. But occasionally the protein is slightly better, and that leads to evolution because a mistake is copied faithfully generation after genera=r"I ~-" ' .' tion. Now, that's what distinguishes the living thing from a crystal. ±° - Really, it's heredity, which a crystal doesn't have. The bacterium and all- living things possess the ability to pass on traits to their decendants. '1'_ But, most important, if a mistake is made, if there is some damage to the DNA, that's passed on, too. That ability to replicate any change is what allows the possibility of evolution. Without that, improvement couldn't be' couldn't be passed on and, therefore, and if detected made or detected , , capitalized on by the whole race of that kind of organism. These mutations" are very important. Although most of them are lethal, some very small " fraction, one in a hundred million, is good. By that mechanism, organisms~ improve. What's incredible in that this'simple organism, which is completely ' reproducing itself and all these toys every half hour, has sex. There are'~, male and female bacteria. They don't need it for reproduction.' They reproduce by getting long, dividing in two, and going on forever. But one . kind of bacteria, called males, or if you prefer, donors, has the remark-. .- able ability to break their chromosome. First of all, if you have a whole . beaker of males you go along producing nothing but malest and if you have another beaker of females, they will produce nothing but females. And, indeed, this maleness or femaleness is one more trait that's specified on-' the DNA. so all the descendents forever will be of the same sex. But if - you mix them all together in a warm room that's nice and quiet and you c• gently shake their flask, they will find each other. And, although there d h eac may be ten billion of them in a small glass of bacteria, they fin other in one minute or somewhat less. The males will form little bridges,' which connect the male and female cell. It's not a sex organ in the senaeq•t it can happen anywhere on the surface. But that bridge, once formed, : allows the donor, the male, to pump DNA through this narrow tube into the cell of the female. Once inside the female, the DNA sits there for a ^1 little whilej but there are scavanging enzymes inside the cell ready for ,c ' a such things. They chew up that DNA pretty fast. occasionally, however, f th e piece of the DNA survives, and then it can be exchanged with a piece o female's chromosome and that corresponding piece of the female chromosome is ejected, ultimately to be chewed up. What results is a female that now iece of male DNA built into her chromosome. This process is called has a p and so thi is a recombinant female (see Figure 6).. Now, if recambination , the part of the chromosome that got built in is the part that specified the ;; i ve•„ enzyme that chews up penicillin, then a previously penicillin-senait ~r J F fesiale4 cai~ noM havd~"proqeny~tAat are •penicillin-resistant. :Ay,.c You can do an amusing experiment. It takes ninety minutes for this;,.; o{;: whole process to occur - for the whole chromosome to go over. If after ten•,; minutes you take the gently rocking flask, put the contents in a Waring;. blender, and break apart all the mating couples, then only ten minutes J„r,,.r worth of DNA has gone over out of the whole ninety minute cycle. If you..,;;-'c find that the progeny are resistant to penicillin,, you can say, "Well, penicillin must have been coded for in the very first part of the chromo-;,t,,., some_." You can do that over again with streptomycin and vitamin B and all,r,'+° the things you can think of. Thus you can establish a map, so you can know where every gene is. Now, we've been running this organism into the ground,:; , for twenty-five years - me'and hundreds of people like me all over the world. Of the five thousand ootential eenea that are on the E. coli chromo- , some, we now know where five hundred of them are.-- So we're about ten percent of the way.. We haven't run it into the ground yet.,; ;r,;, It takes a lot-'of machinery to arrange this kind of sexual transfer.'SIIs There are incredible photographs showing both the male and the female w; - happily dividing and splitting off progeny any time they're engaged in;thia . sexual conjugation. The question is: "Why do they need sex? Why, in evolution, when there was so much competition for efficient, rapid growthp,.' would the cells go to all this extra trouble just to establish a specific ;, mechanism like sex?" The answer is that it must have had an enormous advantage in evolution. And it did. The chances that a lucky mutation r v„' would make this male resistant to penicillin are very small- one in a'r..' s hundred million. The chances that it is also lucky enough to be resistant to streptomycin are about the same order - very unlikely. But if the male -:7' : mates promiscuously with a female, then the resistant male can generate a whole population of resistant femalesi and then the female can go on and mate with another male that in resistant to streptomycin, and another male and learn how to be resistant to aureomycin and so on. in the case of . bacteria, good news can spread very fast by promiscuous sexual contact.,,-, The conclusion is that the function of sex is to communicate _informations and is not at all required for reproduction. In fact rabbits also repro- y- duce asexually.. You can tickle the egg of a rabbit and put it back into ;.~ the uterus and get a new rabbit without requiring sexual contact. Tickling can begin that process artificially. The progeny have genes of the mother f: only and, thus, the offsprings are all identical with the mother. This is,; - real cloning, and it's done routinely with fish. There's a lot of dis- y_ cussion that maybe that kind of research should be forbidden.• It's obviouil" where it's going. ;r. Some bacteria-are%capable of a very much simpler process-; If you put. DNA in their medium, it will just leak into the cell and form recombinants' by the same method. It's a process called transformation, and not all U bacteria do it. Figure 7, titled "Virus at Work," is kind of melodramatic. There are bacterial viruses with incredible engineering. This particular one has a hexagonal head made out of protein packed with DNA, it has a landing gear, hooks to fasten it into whatever it lands in, a hypodermic syringe, and a trigger. This virus is only about one ten thousandth the.; ; size of the bacterium,:and you can see it only under the electron micro , scope. if the virus lands on a bacterium of the right kind, it fixes itself with all this equipment and then the trigger collapses the neck anav . injects DNA so that the whole contents squirt into the cell. Once inside the cell, the viral.DNA tells the cell, "Stop whatever you were doing and-, start making virus pieces." It then begins to manufacture heads, landing gears, neck pieces and more viral DNA. Twenty minutes later the virus J gives a signal, "Let us out'of here," and the bacterium starts to manufac~ ture an.enzyme which digests its own cell wall. That breaks opr-n the bacterium and two hundred new viruses come out.'. The cell is dead,'and sI e ' ' s a remarkable land. It those two hundred go looking for a new place to sequence of events..:sometimee one of these viruses, instead of packaging Fc - i t its own.DNA will package bacterial DNA by mistake. Then when it lande, squirts;bacterial DNA into its new host. ~That's another [, way :r , of transfer- . • . _ • .._ . . ,

rieg,qenetic information, but'it happens`only about one time an,a hundrad'f.y thousand. However, a beaker or a culture of these bacteria can easily ~; contain ten billion individuals, so it happens a lot. Figure 8 shows an ; 4, electron microscope picture of one of these viruses broken open. Its DNA •_ has been scattered all over as you can see. I_t's about one-twentieth the amount of DNA a bacterium has. Up to now, we've been talking about natural ways to transfer genetic information. Now enter Man. There are two new tricks, and those two new tricks, together with what I showed you before, are what's known as genetic engineering. One of those tricks (Figure 9) is very simple. You take two different bacteria, A and 81 you treat them with solvents - for example, polyethylene glycol works extremely well. A and B become fragile and gooey _ at the same time, so that the two cells can fuse together and form a hybrid. Their DNA molecules can then recombine with each other and end up with one whole hybrid chromosome which is sort of a random mixture of the chromosomes of the two parents. Congress hasn't heard about this one yet. As you know, they've been debating a lot about the other kind of recombinant DNA. And they will debate about this, too, because all kinds of novel combina- tions are possible here. It's amusing, when you consider the controversy, ' that this kind of fusion was first performed about fifteen years ago with human cells and mouse cells. You could make a cell that was a hybrid of humans and mice. That lead to the study of human genetics and, now, work : in human genetics is proceeding with cultured human cells just as with bacteria. It's a lot harder, but we have only something like fifty to a- hundred thousand genes. That's really not much more than a bacterium - only ten or_ twenty times more. The difference between us and the bacterium is not our chemical virtuosity# it's the software. We know now where about . two hundred of the human genes are - two hundred of the human genes have been mapped. The rate of progress ie slow, of course. We map about two a month, if you follow the literature, so it's going to be a lonq time before we know where all fifty or a hundred thousand are, and a lot of them ar_e_ going to be subtle and difficult. Finally there's a technique called gene stitching, which is the one° that's been getting all the press. The technique (Figure 10) ie thia: sometimes a bacterium, in addition to its main chromosome, has an extra little one which is called a plasmid that can be gotten out. That plasmid is very often quite a scary object, in the sense that it can contain informa- tion which teaches the cell to be resistant to penicillin, and also to'be resistant to streptomycin, aureomycin, tetracycline, and so forth. Japanese ~ researchers discovered a plasmid that teaches its cell how to be resistant ' to seven or eight antibiotics. That's bad enough. But the real bad news is that this thing goes promiscuously from one cell to another, so that if one bug in a population has it, it very quickly communicates this informa- tion to the others by means of this plasmid. The plasmid can be extracted . by very clever techniques which will be described by our next apeaker, cut ; ~ open, and another piece of DNA from some other place can be presented to, it. Once the molecule has accepted the alien piece of DNA,:it can be resealed. The plassdd now consists of what the bacteria knew in the first piace, plus whatever is on this new piece of DNA. Then, by still another :?" trick, the plasmid can be put back into the bacterium. This remarkable +'trick is possible because DNA from all sources ie chemically identical.. Thu., ths alien DNA can oome from sea urchins or salmon or, indeed, from ~ humans. This means that we can put human DNA inside a bacterium and the f* bacterium might saake whatever the DNA says to make. The dream, of course; -S?' is to make insulin, a drug which ia in short supply'and urgently needed. And, in fact, a group at the University of California Medical Center has a+ succeeded in getting the gene for rat insulin inside of a bacterium.• They ' v = can prove that they've done it, but unfortunately no insulin is coming out r-~c because they don't know how to switch the control mechanism on. They have 4{ to learn how to eay, "Start." They know how to do it in theory, but not yet in practice. I will take any bet anyone wants to offer that within a year they will announce that they know how to say, "Start." The reason tZ~ .t:c they ara`using rat,insulin~instead ol;hus~an insulin is becauee of the' ._• obvious legal and ethical,problems; But once you can do it with rat,'<yo can do it with anything.•,3The technique will be just the same. You will hear in subsequent talks,,,i'm sure, of all the wonderful thinqe that can be ; ,manufactured in this way,that can't be manufactured any other way. There's ' :no serious hope that you can make insulin synthetically on an lndustrial iscaie, but you can steal the gene from a human cell and get a bacterium :.growing !n industrial size fermenters to make lnsulin for you, and a whole : •host of other substances that will be discussed by others. I've taken you part way to a complete description of one'living"thing. " "But we have to go through one more part of the complexity - biosynthetic ;pathways, the sequence of steps by which raw nutrients.are used to make • products useful to the cell. In bacteria, for instance, lactose is broken " down by a lactase enzyme to glucose plus another sugar (Figure 11). Then,~. ~'=by using a series of enzymes, we go from glucose through a series of step_s ': through successive intermediate products. At the end of something like 'eight or nine chemical steps, we end up with products that are useful to Ithe cell. They may be amino acids. They may be DNA or BNA bases. They may be components of the cell wall.. They may be vitamins. Or they may be things which are not useful to the cell, like large amounts of penicillin or large amounts of streptomycin - things that we want to get out of the cell. Or they may be proteins. :(There's single cell protein that you'll '''hear about later.) In the normal course of things, the bacterium manufac- tures all those products for itself. But it doesn't want to make too much ' ~ of anything because that will choke the system and will also be very waste- ' ful. Any bacterium that=wastes its time making things it doesn't need is :going to grow more slowly than some other bacterium that has close control " over itself. If a bacterium is wasteful, it will lose out in evolution. The result is, for example, that this whole chemical process, that is, all ` these eight or nine enzymatic steps, will not be turned on unless there's "'lactose present. It won't be turned on either if there's plenty of the final amino acid product, let's say, that is required. Thus, the amino acid, if present, can turn off this first enzyme so that nothing happens.' There are also other feedback loops in which products at various stages affect what happens at earlier stages. Furthermore, there are a lot of other active pathways. There are five thousand genes, and this process we're talking about occupies about ten genes, so there may be five-hundred pathways. Also, during each one of these processes, the cell makes a great number of intermediate products, which very often are signals that turn on and off other pathways. It's a very complicated, interrelated network of .,processes. The circuit diagram is really complex. ll? It' "' From an industrial point of view, what is this ces a chemical i:factory,in which each enzyme does some very simple thing, like an organic chemist would do. it may acetylate a product to start wit_h, then it may phosphorylate, decarboxylate, traneaminate. You can make a list and you'll find that most simple organic steps take place in the cell some- _ where. Thus, if you want product PZ, for some reason, (eay, for example, :, it's a`precuraor for an amino acid, or maybe a'chemical that's useful for some industrial process), you begin by mutating a large population of these cells until you get some that don't make amino acid any more. You keep them alive by feeding them amino acid. The ones that require amino acid `are the ones you damaged at the desired place. The chances are one out of eight or nine that you've damaged them at the place you want, and, there- fore, the product you want is going to pile up in large amounts.. By this ; process, you can block any pathway you understand and get any product you•' :;want in large aswunts._ It you're not satisfied with that, the next thing ; -. h, you do is mass up the oontrol mechanism so that the thing runetopen loop -. so it runs flat out. If you're still not happy with how much of the pro- ` `' duct you have, you can expose the cell to various chemicals that further alter the genetic information in the cell. These chemicals cause certain genes to dislocate themselves so you can get two copies or three copies or : even four aopies of the genes that specify all these enzymes. You can.. '~11. WON

multiply your yield by even more. Bu!'thereis'still`another trick.(y If you manage to put these genes on one of those little extra chromosomes,.the plasmid we discussed earlier, there are tricks for multiplying those chromo- somes, so that there may be one hundred or two hundred of them all alike. The world's record is one cell turning something like fifty to seventy percent of a total protein as one product. The record with penicillin is overwhelming. When penicillin was first discovered, the bug was making something like one unit of penicillin per milliliter. Now industrially s., competitive organisms are making ten to thirty thousand units per milli- liter. liter. Comparable improvements.are expected for other processes. An organism can be engineered to perform any sequence of events an organic chemist can do and some that no organic chemist would undertake, by asking the organism to do any list of simple side group.manipulations one at a time. Say, you find a bug that grows very well on something cheap and another bug that grows on something very expensivel but the bug with the expensive taste makes lots of penicillin. You can combine those two bugs by one of those trickst conjugation, transformation, transduction, or by one of the artifical gene stitching techniques or by cell fusion. As a result you get the beat of both worlds - a bug that grows fast on something cheap and which also has the ability to make a lot of penicillin. Genetic engineer- ing is essentially the combination of genetic information using these genetic manipulations to make super organisms.. We choose the bugs because_ they use particular processes to make particular productst to leach metals from ore, to clean up pollution, to produce large amounts of protein that .~ have a good balance of amino acids for nutrition purposes. (You'll hear about all of these applications later in the meeting.) Why do we call it "engineering7" It was a science until a short time ago because you didn't know what the outcome was going to be. Now it's become engineering because - we know how to do it, we have a table telling us where all the genes are. , For e_xample, we know if we let two particular bugs mate for thirteen min- r a certain gene ' utes but not for fifteen minutes that they will transfe that we want. Thus, if you come with a shopping list into any laboratory;;- engaged in this business and say, "I want a bug that will grow at 306 but not at 405, that will not grow without vitamin B, that will make lots of ;. tetracycline, that will not do this, but will do that," within a matter of a month or two - not longer - a competent lab can manufacture a bug for you. Now unfortunately, it will almost always be an E. coli because that's the only one we can manipulate this well. There are a few othera, such as Bacillus subtilis, a soil organism, that we know quite well. But E. coli was chosen es a-target of this conspiracy to run it into the ground because it's a harmless organism, because it grows fast, and because it will live : on almost anythinq. It's a natural inhabitant of the human intestine, constitutes a substantial portion of human stool. It's all over the plece, all around us, and is more or less harmless. What couldn't be anticipated was that all theae tricks apply exactly to all other bugs. Now if someone says, "We're extremely interested in this pazticular bug," you can esti- mate how much effort it will take to understand it as well as we understand' E. coli. Making genetic maps of other organisma is completely feasible. ; t's an engineering task in the sense that we know what to do and we know it'a qoing to work. It's a lot of work, but it's a definite project. _ ~ Now I'm going to shift gears in the last few minutes and tell you some 3 of the hard realities. What I've been'telling you about until now are the the generalizations, the logic. I haven't really told you lovely ideee, :'.,how you do experiments to prove these things.,.: It's a lot of work and we for color ' lling things or lookin always end up eountinq colonies or smeg : changes, etc. Because it's so much work, we have to aski "Can we really''` ics and chemistry or are there going expect to explain everything with phys , I would bn delighted if it came out to be some new principles7" Frankly, } either way. Nothing would make me happier than to find some now principle, y,; could say, "Aha~ 's is. But it would be L so that all of us . That what life '" Yeibvious consequence of ph "Life reall is an o ica ust as pleasant to sa ,.y, and chemistry and on every planet that'e',ttiendly enough, life rill devolop; It has to." Either result I will find philosophically very exciting. But you've got to believe that rational laboratory.:methods are going to work in order to be willing to get up in the morning and go to the lab every day. ~ The working assumption is that conventional experiments and conventionalr,- . ~sciences are going to work. But there's a lot of work to be done. Realiz- ing this, I set out ten years ago to automate the more tedious, repetitive parts of this work. Figure 12 shows part of an experiment which lead to a ;.'recent Ph.D. thesis in my lab in which a student-examined in detail a million and a half colonies. Here they are. These colonies were, first of ` all, laid down by a special seeding machine that puts down one cell and only one in specified places. These colonies are not at random. They're in a regular lattice pattern. We take time-lapse pictures of these colon= ies as they grow exposing them to different temperatures, drugs, viruses, nutrients - to all kinds of things. And we challenge them. "Can you do this, can you do that? Will this kill you, will that kill you?" I'm talking about one and a half million colonies, and for each colony there ` may be thirty pictures.~ Nobody can look at all of those tens of millions'- of photographs. Therefore, we have a complicated television camera con- '_ nected to a large computer, and it does all the looking. It_ measures everything,: In fact, each of these colonies is represented in the computer ' by something like a hundred numbers. We measure a hundred parameters, such `as color, diameter, surface texture, shape of the perimeter. It's a complicated mathematical description. The result of all of that is that the computer says, "AhaS I found what you want on dish number 34," and draws a picture of dish number 34 full-scale. Then the student puts the dish on top of the map, and the computer says, "Get me that colony." So the student, with a toothpick, recovers that colony. Then the computer says that the next good one is dish 51 - "Here's the first mutant, and here's the second mutant," and so on. In this process, there are no tables of numbers. It's simply a mapping game. But retrieving turned out to be the main moron job in the student's work, and_ it took a lot of time. So we made a thing we call "The Hand" (Figure 13). The Hand is a four inch block.: of aluminum that has four hundred fingers sticking out of it. When we find a colony that's interesting, we_ move it to a standard index position, and The Hand stabs the colony with a slender quartz rod and picks up some of the cells - maybe a few thousand cells, because the colony is sticky. After the hand has done that four hundred times, it then can print a replica of those four hundred colonies on some fresh agar and you can do some new things with it. That's one sort of harvesting machine that we built. Success in these areas led us to the decision to build a big machine.' Figure 14 shows a gadget called The Dumb Waiter. It's big by university standards. You can see there'e even a staircase. The machine consists of a lot of glass trays wtiich circulate inside of it. It's a sterile incu- bator inside. There are a lot of little gadgets for photographing, and The Hand is mounted on it. It also has an inoculator, and any kind of manipu- lating devise you wish to add. There's a picture window down at the bottom so that the biologist can scrutinize his experiment as it goes by and see if it's been wiped out by mold that day so there's no point in going on, or if somebody forgot.to put the glucose into the agar. It's a big expensive . machine, and so we don't want to use it as an incubator only. We keep a lot of experiments going by unloading the Dumb Waiter back into the machine shown in Figure 15. It sits on a little hover craft so that one very frail person can push this into a warm room or a cold room to incubate off line. ~ 'Figure 16 shows'a photo showing some of the dishes. To avoid mold we- take enormous care about sterility. This is a super clean room with lamin--- ar flow and fancy filters and all those things you know about. Figure'17 shows big sheets of glass carrying agar. These`ari u:ed in " the dumb waiter. They are about one meter long. D Fveyt y~ : E a' x'+~t 13T.~y , * / M!r"~EL~~i` eY'n~.^ -L.~"J"~ ~`i ....._.

Figure'19 shows what a hundred thousand colonies looks like,'and what ~,:~ my`student did once each week for the required number of weeks. It really ~f '_ isn't at all bad because the machine does the tedious-'part. '• r?a Figure 19 shows what some of the colonies look like. The'coloniee in+` this photo were from an experiment to try to do automated medical diagno-•" sis. This was the game: the urine of twelve patients who had urinary infections at the Stanford Hospital were shown to the machine. After being taught what these twelve organisms looked like, the machine was asked to identify a mixed sample or a sample of unknowns. It got it right so that the error rate was less than one out of a thousand unknown colonies. If ' you're really sick at a real hospital and your urine goes to a real labors- _ tory, it's pretty good if they get it right sixty percent of the time. So this is a lot better. It's much too expensive and elaborate for any real hospital to afford, however, and there are a lot of good simple methods being developedi so I doubt if this is a serious application. But it could do well at pollution monitoring, or other tasks which don't require immedi-~ ate interaction with a patient. Anyway, here are some of the things that the machine had to look at. These were some of the things that it could learn to tell apart. It took black and white pictures in five different,`„ colors of light, finding about a hundred parameters for each colony. In the last couple of minutes I'll tell you what we're working on now, and that's the cancer problem. We've learned how to grow human cells in our machine. They grow in just the same way as the bacterium, but it's a lot harder. Nutritional requirements,'everything, is more difficult. In Figure 20 you can see some colonies of Chinese hamster ovary cells. What's remarkable is that after a week or two you get a sort of a "fried egg" colony (Figure 21). It's mu, h more complicated than the bacterial colony. Then in just a few hours the cells begin to run away from the yoke of this fried egg and form a sort of a cart-wheel. A few hours later you get a sort of a lace doily effect (Figure 22). It's as if these cells are trying to make some kind of a structure. It's as if they remembered that they belonged to a particular organ and are in the process of morphogenesis,,o making a complex shape. They move around, they recognize each other, they stop moving. Some of the cells starve and don't do anything else. Others go wandering around looking for more nutrition. They're very complicated celis. We expect if we damage the cells, through mutagens which damage the DNA and which are the substances, by and large, which cause cancer, we will see , changes in these patterns, because these patterns depend upon a tremendous number of genes. Furthermore, even substances which don't cause mutations may poison some of these steps, and so may be analogous to some of the changes that cause birth defects. So we think we may have a rapid labora- _ tory scale method for detecting carcinogens and birth defect agents, as well as simple mutagens. + ` We also thought we might be able to detect human hormones and, in general, substances that are present in extremely low concentrations f, There's a detectable and measurable difference between the appearance of. , the colonies growing in normal medium, a very low level of inaulin, and asa very low level of male sex hormone. The computer recognizes these differ-' ences very easily, and can tell us how much we've got and which of these ~ substances we've got.. So we've got an assay, which is a very important = first step in trying to find cells which secrete things that are important to us for whatever reason. • This talk is a summary of so'much information that'I won't try to o summarize it. Obviously what I've,tried to show you are the fundamental.: principles. I have a long list of applications which I:will not repeat because I've talked a long time and because the'following speakers will ' tell you some of the more professional details about how you really do j these things. There are many, many applications. But in addition to all y the things you will hear about - what can be done by bacteria and mold and k -yeast, for example - I think the future is very likely going to see us growing plant cells and, in fact, we're trying to do that in the Dumb ~ 14. 9W ~z000t . W; f l T+i z J3uf s~~'rI ~ Yi • F::jiedC.ki 1~:,~ »~3! 7.c..:,i..µ fY. ~. s t- Naiter machine. "I! wa sycceed; - we' 1X be able to ioprove czop yields for , .:. agriculturally important crops, and to develop strains that are resistant to rust or resistant tod droughts, an so on. Finally, I think you will see human cells and other animallli i cei growngn machines of this kind to improve yields of products that those cells can make. What I've shown is ¢ort of an academic university instrumentation approach to doing research into fundamental principl Ahi di es. macneesgned for industrial productiori or for industrial development of strains would probably look quite dif-., , ferent. Thank you very much es`r;} ju. - .'F: " ;ca J'J, Iz ?rt~,: 3 J,~ ~ 1.c.,.l"z4T+. W. trS •iS' st .~ r 2 S~r~r a-s y trr.•. 7 - pl?[~ t- .ta t -t t rt. ~. t...I i -3 ~.5 c 4~ ~' 2<m t s.• rt fl~,~`i f+ ra.~; 1 ~. a4„ x?hf 41, .i:- N y7y~.~ . r 1r71;W, yt v eN*' t.{ aY.zrj tt ~~r #uf}r r t c_ r .7 c 7~ Cf.••_ sNf fJf..F. ~ A~~ - ~ 3 . - ~ r r ,. s.4 k# '.tt' r_~s~ca ~: ~Pv3 haG r I, i L ht, v.` ta €7 _ 1~7t~d 7 4 = x. t7la6 Y t.~#Er P n,0ik>b~ C` r.a ic .~ y t R , .: ..,,;-f r4ar~'1.... i .TF r~,i i:~ _~. riFr~ r ~-. fUFLd J.c te.ze~ r' rr.: aIV cg so 3 j•ss iS:- s7"~.L.iC41Cz 001 ; fG >~ 4 v3 ~. ;i r t rF-.s' ;.,It.1~x - ~ r ~~rg;e:'Yy.• -~az i~yrte~tr^+sx:,~~ 5 yc~ rSt.a~i, ~;nw ~. r ~tiy u~-~?• "1 ~~.rc~G+bX- ~cF ti*~ j~~1. at~ -fC K'P~,-1 S..ff. FF;~un /•;t 1N: ' ws s~S ~r% K"ns+k .3ef~f #t~k3~)} ?ax• x?~z r~avF ~~x~.' aws x;St ::_ u;r_ti,i l..~t'?• ?~ :f. ,- i2.'?r_xjt 3fs r`'":i ~]] t~; ?xe c.: r a~ .aiTT s ~. Crl iF.{fit- a~ U r ~7j YK?~h *ftiiu f~tc.Fi~ ~a c ~rr >1s, x'FK ` V :i1 fi }t °o) ,~ _ w b iny . f~gr ri~cr~~~T st 4,15. :r tt.:iF~ •-!' , yfSl r:,RO),.f>

~ l

a