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G ,~i ~a'.~.'li iL +7..?~&~t ~.. :. ~-~ a~1ISJ`~ . ..a+izv ! ~ff r- ; t ~ W ^5 tQ. - . . _ , _ .t. . .. ,:., _ . ~' b A Sujiyl.~~ ~ J~3,,~~ [;?t ? LA.L ~!t uc C ttJ~~, ~~ ai $~., ~o~ r~ptuu 1a;,dq r 4.,=7,.,~} ~ MICROBIAL GENETICS AND THE, FUTURE ~Z'Sr,., ~;:~.I:Z ~~ 3~St~ '; . OF THE PHAR>,'KACEUTICAL INDUSTRY ~ ~3o Ronald E Cape, -Ph D. Ysy ~~ ,t v ~3y"F ~x -Cetus Corporation Berkeley, California 94710 . s:)6, 1;yl ~[G .:l~C'~ ~. L-y. _ y' + ~ {f$4Y.~y~4I:; .iL3 a ~ L Presented at the 4fl CHEMICAL SOCIETY 3.. MEETING ::"New York City, New York 3, ,April 6,, 1976 ..,J <. .."~~ . TQ I:O~~c . ^ . t .. . ..._ .~ J _ ..> `r.

C r ~ un,P, ~ ~''a t. . - _ . ~ ~ ~f F f^~.~ + . ~~J a There are a numbervof themes I intend to discuss simultaneously in - this paper. :~z Ff ~ n K e~~; This report represents perhaps the hundredth time many of you have heard some- ~ ~ one pontificate about the need for a more enlightened approach to the search~~~ ~for new microorganisms for the fermentation industry. I will review for ~s you, r x; . somewhat sketchily, some of our mutual understandings of what the genetic issues ~..~; are, how they've been elucidated and applied in academic institutions and..in ndustry, and what the accumulated body of knowledge makes it possible eitber v 41 Fa ,~ o do today, or to contemplate and plan today for doing in the years to come. he genetic concepts, of course, aren't new; they were addressed by Darwin and 1 -`<by Mendel more than a hundred years ago. First is the observation that biologi- ,gb"r"_cal organisms, by and large, breed true -'Escherichia coli give rise to more 1'.{; ~ coli, and elephants to elephants. `The concept of species is important here = with a few marginal exceptions like mules (which are sterile) and. beefalo (which re not), breeding takes place only within a species. A bacterium.could not be „r.ne recipient ot genetic information from a fruit fly. ~(That is, not until recent- 1y, but tnat gets ahead of the story. ) Second, within a species, there is a certain amount of variation. .Mendel showed ` ,~ y~ that breeding among the variants produced mixtures, combinations,`in the off- ' ~ '~ ~~ ~ 4Zspring, of characteristics of the parents, and did it in predictable ways. :: In •t spectsof the ~ . variat ~ r l h t d ~~~~~ ,, ~ sa h e ~ n e . me p enomenon Years ago one referred to dominant and recess recessive genes ~ {Y ¢,w: today, we're more likely to talk about defective enzymes, blocked pathways and a , ` •.~ . ~~~ the base changes and genetic code ramifications of deletions and frameshifts ~ ~' Bu t again, I'm getting ahead of myself. ~~ * '~w third and last classical concept I want to mention is selection. Darwin „ talked of "natural selection." ::Survival of the fittest and so forth. ": The point ' ;~s x; r is that some changes help the organism, and some hurt it. I S.if' . . . . . . Of course, all of this says nothing about the mechanisms responsible for the var ~ iation. When I was in high school 30 years ago, we were taught that genes were ~ abstract concepts only, and workers were only beginning to argue about the rela Y,tive merits of proteins and nucleic acids as informational macromolecules. Now ~~ ~'~_' we know, for example, that variation is an expression of mutations permanent F~,'° ~changes in DNA. ~ 'c" F3'~. +J is remarkable what workers have accomplished (from Mendel over one hundred t ears ago to Beadle and Tatuml and Lederberg2 35 years ago) without a knowled e~ ~r g li i f the underlying molecular biology. In the pharmaceutical industry, a decade before Watson and Crick3, tremendous advances were being made in penicillin yields, . ~ ~ , ased on the observation of occasional variations in nature against a backdrop of ' biological organisms tending to breed true. So at first, penicillin yield improve- ~~' ments came from finding better natural varieties of the mold Penicillium - dif- ferent wild types, as they're called. . ~:._ :In the Penicillium case, however, chemical and irradiation mutational programs ~4~: began to supplant the search for naturally occurring, wild isolates, and as yields improved, a series of other, less desirable, attributes began to accumu- late. And so today, to dismiss a complex matter in a very few words, the pro- duction strains of Penicillium, havin N~ g yields many orders of magnitude above - their ancestors, are extremely fastidious and require careful coddling to "do `~ their thing." ~ Figure 1 illustrates a recent industrial lineage of Penicillium strains which represent a genealogy of mutational offspririg. Little is known of the specific ~tY ,~i Y ~ ~~ '~ ~ ~.* w rc-' yv 3 3 • ~! l:fa~yl{G~.~~'~~ S N k~.r ~. X aig 419 f Yast •J

T,:....-. ~~ . . . _. ... . ~' ' nature of each favorable mutation (i.e., mutations resulting in.greater.penicil- - ___ lin production),=to say nothing of what other unseen or unknown mutations`have ..... accumulated over the years.-~-As the geneticists will_tell you, if you don''t;select _ -for a trait,-you generally don't know it's there.,;_:But it's clear that what we,~ i -~.get at the end is a highly crippled organism.,-:,It wi11 grow well only in ~care K .. ~ ::. . :..: . w ;fully-controlled conditions -..it probably couldn't make it competitively iri the ~?~outside world.W~~`~To go back 'to the concept of. selection,` Darwin's survival "o£ the '-'fittest principle never wouldyhave,favored today's production Penicillium organ- isms.` Natural selection didn't select these bugs :.the workers in the drug com- panies and universities did the selection.`=Let's call it."unnatural" selection. (Let's also call it a pain in the netk!) Utilizing a variety of'mutagenic treat- ments `the_action of-which we now understand-somewhat,`workers permanently'alter ,. ~. ~ ~ t- k the -genetic'material the coded nucleotide sequence in the DNA .- 'in such a'- way ~~that some ofthesurvivirig organisms are demonstrably different-,Let me remind _. _s ~ that;''in"''' you here,theabsence of furthermutagenesis these different organism "breed true:".~.~If rthey.didn't there'd be no purpose to.the proceduret i:But in}` „most industrial situations, the characteristic.which makes them mutants also:,; .,makes them cripples in the competitive struggle to survive and propagate in ~nature ::So the'desired mutants don' t show themselves in Darwinian selection ='ssituations '"This is in contrast to the so-called "elegant" genetic experiments ton E. coli that have taught us so much about what the newspapers like to call "the secret of life. "~~In 'the latter experiments, Nobel Prize-winning professors were =~! ~able to utilize natural.selection -.the experiments familiar to you all with auxo- ~:4-:, ~;~trophs and selective media. : But, few if any such experiments have been reported ., r~ ;:rrtin which, say,'an already damaged Penicillium (by way of example) can be furthe amaged by mutation to,allow mankind to squeeze 20% more penicillin out of it at . .~:~each mutational step,=and then proceed further to win out, to overgrow its parents "i ,siblings;-and.ts'far healthier.ancestors that may have managed to reenter the ; ,_ M.. . , . r r A~Lfrt ~~[C ~ rt rt i* r~ t1 3~ 2J ;11 S~l.rs r~~ :_ 'w~~«:~j S.~lf 1 picture r F~t s.ri!1 w~i'S~ ~' ~ ~~`w ~ t . ~t. ~ y industrial microbiologists have had to resort to a far more laborious and :; ~ak````inelegant strategy; that is, growing from a single cell genetically pure cu1- S,,. ~ ,~,''tures of mutant progeny and testing each genetically pure colony for, say, anti- _ ` bioticproduction. Now, antibiotic production is a trait which, although import- ant to patients and to drug company employees and stockholders, is, as we've ust pointed out, of damned little survival value to that Penicillium mutant in _. ' ~:,~the shake flask.-. Add,to this the fact that frequency of favorable mutations '(mutations.favorable to man,'not to the organism) is very low...;This means screen= .,-ing very large numbers of colonies.'.`-That means large numbers of flasks and very many antibiotic assays! . It's very'laborious and slow - but the industry has ta ~~ accomplished heroics with it over the past 30 years. Many orders of magnitude -wincrease in yield, in many cases A~to' e, o b FS ~' s=f y~~~~~ _yd j~7at7~T5 I~3 ~~~ -Needless to say, any means of automating this process, or anything else to com- press time in strain yield improvement, is a big help, and several programs are under way, including those at our company, to do this. ... .._ ".... .I'" ~'sdl Tn addition to improvements in industrial production yield - very important to return on capital investment - several other lines of related research have been carried out by the drug industry. In the main, these other programs relate to the discovery of new antibiotics and the understanding of their metabolisms in the producing organism, and their mode of action in the organism they're intended C -to combat. •I can only devote a word of mention to the enormous world-wide screen- -=-.ing efforts which have resulted in the discovery of many thousands of new com- _:` .pounds with biological activity, and the extensive, expensive, long-term toxico- logical and clinical trials though which so many such compounds were taken and .which so few survived. And the mutational and precursor feeding studies which '~'

produced new antibiotics. Hybrimycins and mutamicins are excellent examples. :Or the structural and pathway studies which made possible new.anti'bioti'cs by = feeding fermentation organisms with special precursors, for example with pyrro- ' °linitrin or metabolic inhibitors such as sulfa drugs to give 6-demethyl.tetra- ' ';±_cyclines from tetracycline producers.•`Or semi-synthetic antibiotics,-like ampi- rcillin and amoxicillin come to mind._:And one has to mention the elegant elucida tion of the biosynthetic pathway df.tetracycline by McCormick4.and his group. r`r~'~~` Consider Figure 2 but please don't `try to study it in detail ?:''r draw your atten- ,.-..~~ n: tion to it merely to illustrate the complexity of the pathway and to..stress that "` "` this kind of detailed pathway knowledge is exceptional in the antibiotic field. But as pointed out, this knowledge did pay off ) ". V ~.N ~ ~ ., ., ..• . . ,,.. .. ;:'Antibiotics have also contributed some highly heuristic insights regarding fun- ~ I damental discoveries in molecular biology : There is, for example,streptomycin ~ which causes mistranslationby ribosomes.- Then there is puromycin;`which struc- . ?aurally resembles transfer RNA, or actinomycin D; which binds to DNA.;.And, of course, the 6-lactam antibiotics which interfere with bacterial cell wall syn- thesis ':More can be done, and indeed more has been done than has been published. `rN °l t . •;'. . ...-. .. . _ . . , _. . . .wd.d'_ ...~.:4.,~ : So industry does have a solid record of accomplishment, and society,r'and of course :;. the companies, have greatly benefitted from the work. lt' ;aBut what remains to be done by the drug industry is to fully'exploit what has a•;~r,been discovered in molecular biology and genetics during the last 30 years.-:~a, ~:;r}:=; 1 '.Flemings discovered penicillin in 1929, Beadle and Tatuml elucidated the "one gene - rone enzyme".concept in a mold well over 30 years ago, and Pontecorvo6 studied para- ~`'~sexuality in Penicillium in the 50's. -=: Yet still, the drug industry uses mainly .4:F * •random, blind mutation and selection and simple empiricism, in looking for new 4h s;,a;, ~ Y.microorganisms.•.There has been rational screening and directed selection in v,the more research-oriented companies, but the managements have generally dis--. :couraged publication of the successes. Pontecorvo7., in fact, publically and bitterly scolded industry microbiologists for this state of affairs about a year and a half ago. '.Some exceptions do exist. For example, the use by Elander8 of =hd a stable diploid Penicillium strain synthesizing greater-than-parental yields 4' :.. 1 La^-'+ai ~ ~ jG1j`A~ k str~~k~~,V of penicillin. OR''~ r z. s ,.: . • •. I think there'are'valid reasons for what has happened, and what`has"failed to ` happen.;'But I won't go into them. I would prefer to mention a few of the high- -;.=lights of molecular biology since World War II, and then to get specific about ., prhat are the here-and-now applications which await only the commitment of indus- ~ e'• ~ ~.L r ,'"Y `.. exploit them y S,. try to ~i The discovery, by Lederberg2 and others, of sexuality in bacteria; made it possi- ble to perform matings or crosses between different parental types of these lower organisms - something which wasn't conceivable when it was felt that they propa- gated only by asexual division. ~~`r • ~ ~¢' t1-' r? These single-celled or other simple organisms can generate new DNA sequences (what geneticists call genotypes) in two ways - by mutation, and by the mixing of parental DNA in sexual mating, by the process we call recombination. Recom- bination was the phenomenon'which Mendel was studying when he looked at crosses between different kinds of sweet peas. Mutation and recombination, as Hopwood9 `•has pointed out, play complementary roles as mechanisms for the generation of new genotypes - mutation is a divergent process.whereas recombination is conver-, gent. Mutation and recombination work in virtually every natural biological system to create maximum diversity, in the fewest number of generations. This provides :` t:1J.J(a`

the best mix for natural selection to work on ' Yet industry has, by and large, :j'ignored recombination, at least in its publications." Partly it's because nuta ~~ : "'tion alone haspaid off'so well "partl p due to the difficulty -`the time ~`'ahfwrn otP rha nd te cost -'to _okig uM thelaboraory Procedures to actually observe Wand y ' ~'studr`ecombination in 'each new species --iBut I am suggesting much wider use `~ ,.,.of recombination'in industrial organisms;'and I am~suggesting it principal y l as~ ; a Tteans, ~s T''Ve , ju'St said;` of more quickly introdiiti it. th ~ ucnq more varaonnae population ~`nds." But we all know that recombination can pay even greater divide' Ka dombinatldri 1 as also ~een~an' ~indi`sperrsible tool' in gen`etically mapping' the 'z , it . ~: se q organisms whomaps have, .in turn, revealed many 'of 'the "secrets' of life" we~ ' •7kh..! . .EJr .. . . . talked about earlisr By mapping, we- iaean finding the'relative locations on-"3 the chromosome of the'DNA sequences,`or genes,' coding`for specific enzymes:`'xLet me show youI two figures~just as an index'of tlie "relative'amount of work which ~ . _ ,...,. has gone 'into" the'`study-'of the economically."useless." E.._;ooli on the one." hand, ~ ,'and a streptomycete more closely related to antibiotic-producing niicroorganisms~j roh^otr h""` Fst, stto n teheandir note therepmycete (Figure 3)."As you know,'each labelled point on`theinap reflects a'certain amount of work'and knowledge'about' the genetics and biochemical pathways of the'organism.'''"This streptomycete'is the :. most extensively mapped. 'Most others, including those which make important anti- a;~ biotics, `are' much less well understood. `Now consider E. coli (Figure 4)~The r message is clear The genetics of industrial microorganisms has a lot of`catehing~ ~t'c4 up to do S A `~1'~C~4Z°~f~'~~2 C~ ,.~ShcR"Yt?~+~~x„x .. U~- ;' But if industry would commit to playing this game of catch-up the benefits of f, a a~ ~S.n~.... .. .. ._ . .. .. . . : i~",';, such genetic_knowledge would be considerable.` Table I summarizes some of these. ,;..To name only a few benefits, first there is the greatly increased variability ~~ ,$;already mentioned. Then we could look forward to coordinated work between gene- tics and biochemistry to further elucidate the biosynthetic pathways for anti- biotics: ` And `theri we shoulcf ~probe the con1rol° mechaiiisms' governing' these path-_ ways - operons,. inducers, regulators, repressors,'*and so on.`I don't want to underestimatethe difficulties here. 'Antibiotics are. not like the sugars and ''`amino acids whoserpathways JacoblO, Monod", Amesli, Yanofsky12 , and many others studiEd to show how cells control their metabolism ".`intibiotics`are secondary °mtakiolites; frequeritlo mae unt gowiis'so ey nt evendilrtl~mehow restricted by theT ,~environment.'xAnd when'you get down to it, we don't even really ally understand w~`A ' ` Still an f th hf not organisms make `an£ibiotic. undtdi o s ersanngeow, i the ~ ,.: .. of-antibiotic biosynthesis-will permit rational intervention in control and other mechanisms in many ways, to improve yields. Such intervention enabled molecular bi&vgists-to-iricrease'enormously cellular productiori of eniymes such astrypto- .,. : phan synthetase or S-galactosidase.`-'.As another kind of exploitation of genetic~ ._. insight,"as werlearn'more.about genetic map loctios of thclld "t ane so-aehot spos," e~ different for each mutagen, wcan vary the use of mutagenic agents more intelli- ' gently. At the same time as pathway knowledge grows, more effective"use'of blocked mutants can also lead to greater ease of us ting selected precursor feeds to make ~'-.1new desired antibiotics.'~'Recombination may also be able to do this directly by' " matings, or crosses, in which, simplest case, some single enzymatic step is added to a metabolic sequence, now allowing a chlorination, a methylation, or ~ .i _. what have you. ;} Lastly,in thisrbrief survey, recombination systems permit a way to directly r" combat that old bug-a-boo - the sickliness of the production strain as a resuJ.t of accumulated genetic damage over many generations. Backcrosses to healthier parents can be'expected to alleviate the problem somewhat. And you can think of the ideal industrial microorganism - optimized for rapid production on cheap .medium and exhibiting no problems in scaling up from flask to ~` production scale i - organisms into.which any antibiotic genes you want can be crossed. -The whole idea is very attractive. ~.;r-

`J$ tr..') ~ " But when we read today of "recombinantDNA. we are addressing a remarkable new ~,...._ ; capability. ~ Thanks to the work of Cohen1.3, -and Boyerl4 , ,and others, using some - enzyme tricks to cut and patch DNA, a new kind of recombination has been achieved, in which genetic material has been exchanged across species barriers. The signi- : ficance of this at first is hard to grasp.+ But remember the so-ealled impossi- ~ bility of doing this is,what defines a species, especially.in higher organisms. .. . . .. Nature has developed ways to prevent one species from mating with another. In g~ higher organisms the sperm of one species simply will not fertilize~the~egof , : _.. t .. .And even among single-cell organisms, these barriers.have been hard to -i another. ~ . .,. .._ _ ._ . .. -,.. r cross '~.Now, in its ultimate expression, molecular biology has broken these z~ ` barriers. ~~.:";~ Because at's ~so astonishing, I'll review briefly 'what is.done. a.; 1t~ ~ ~: ...v41a'"•~J. ... ... . ._ _ . ..-- .,.,.. .. ..... ii r Ironically,,thevery enzymesyme.s thoughtto be involved in ma~. ntaining species bar- ... ' riers:are among those employed.It turns out that a so-called,restriction endo- '" ~ ;; nuclease called Eco Rl cleaves DNA in such a way that leaves "stzcky"ends." <:This? w .. s, . _. , is.because it cleaves at an axis of symmetry - a palindrome where the-nucleotide `; _ ~ base sequences read the same on both strands in the 5',_to 3'_direction...`Figure ymmetry._:Figure 6 shows how 5_shows the principleof• palindrome, the axis of s ~ A:.•-1:... ..._ ... . ~,applying this to the double stranded DNA molecule creates a series of comple- ~~ ...:_ ° ~. mentary overlapping, single strand ends -a sort of ~ mortise and,tenon.situation ;, . . , _ ` These are used to "stitch".different pieces of DNA together by_the~hydrogen bond- g between complementary bases. Very briefly, the same kinds of sticky ends_. ';(Figure 7) are formed in the DNA for insertion, and the DNA in the recipient, ~' and then,,very rarely, but sometimes, the pieces come together,,,the way you want :them,.=::Then another'enzyme, a ligase, is used to supplement the hydrogen bonds between'the bases with covalent bonds in the sugar phosphate backbones. The 1 DNA is stitched together again Lt , ~ 'r r~l^Si 5.4i~'h~1roJ+!'` A7. - .,Sofar.Z've described what might be dismissed as merely a biochemical trick. But ,;if the recipient DNA is what is called a vector, or a vehicle - a,piece of DNA like a virus or a plasmid which has the capability of entering the biologic ,.,-t .. . then.it will reproduce. system like a bacterial cell and of functioning there -. . :;s ti,F`'and amplify the stitched DNA along with itself. The stitched DNA "breeds true!" . . ..._ .. . .. ~.. `This has been successfully done and the stitched•DNA,has expressed itself bio- ; by making messenger RNA and in some cases protein, in the new unrelated ...,~ : _ . . . ... . . . .. host ~;(Plasmids, by the way are small pieces on non-chromosomalDNA, which .,, :replicate and transmit characteristics to the progeny of certain bacteria ). ~ y a' !tthl3rr±g :.~.., -:J . 6 r t t bkthtf kll abt th In et I h aeoe so secy, but mos o younow aouis ,n ayven, ~. ~~tfi~4y r ~,.. ~ _ ' :2.. .. , 13 recommend Stanley Cohen s article in Scientific American of July, 1975,..' The ;;techniqueis called, variously, gene manipulation, gene insertion, molecular ' . ~ .~~ cloning, plasmid engineering, DNA stitching. .Most.frequently, it is now referred " " ato as recombinant DNA. J ~.. <.:<~ . r'Y:'f I C'!. .••"~'~ What does this mean to the drug industry? First, because it is a recombinational technique, DNA stitching represents one more way to accomplish the various objec-. tives listed in Table I. But DNA stitching will have greater precision - typic ally small pieces of DNA are used about which detailed information can be inferred.-~4*1~ Thus the transfer of specific, single enzyme capabilities, for example, to try to ~A make modified new antibiotics as just described, will be possible with more pre- 0 cise control. Let me remind you that plasmids and viruses multiply much more than the bacterial chromosome. So you can think of gene dosage effects, say, for yield improvement. Let's imagine a synthetic sequence (Figure 8).. Here are eight enzymes, under some kind of control. _If they are found to be controlled z in some coordinated fashion, most likely they are contiguous genes,and mapping will reveal it.-. Such coordinated, controlled genes are called operons. The J 11 entire operon, being a continuous multigene stretch of DNA, might be stitched . .. =~:~ . -, ~ ... _ .. N

.'into a plasmid,,,with great.amplification and control ramifications .- And of coure . _ . ... :...._ .._. . ...- - "~~~-`?~ ', the recipient organism need not even be of the same_ species -=, Cohen~'s plasmid• ~ " -sZ`4PSC101 seems to be pretty ubiquitous, »by _way -of example;But if the convenient- 'geography and-;control-ramifications of an-operon are not found'to exist;or cannot ' be created by geneticengineering,we can stillenvisage the followin 'I g n all ' -•." . - ~ _~ . ~]ikelihood one oftheseentiteps istherateiiititIt is reason ,__ ;zymac- s_,- _ _mng .sep. :•.- "able to find that enzyme,~stitch its gene.into a;plasmid, and amplify it ouE'= Then a new.- engyme;;,would~_become rate limiting.."-The " Yprocesscould berepeated, and production of the antibiotic rationally^increased ,' ._ wise.:wUndoubtedl some model like this consecutive alteration of a'series, _ _ . - . , . . . , _ . . ~ of knzo ~~ explains iuccess already"obtainl ~muoational programs. Think o~T ,__ .. a bac ~~, a~ t eY p icili n genealogy chart-, ~ ~E chf- pr vement meant "something happened " But we never bothered to find out what that "something" was in each ~ r, h k ~ case ~~~~'~ ~~~ ~ ,~ ~ ~`{+'SCJR rn'-.i7• . ~- . r_t!tF;~wJ'4 1.41::"~::.:.i:i" ifF`.'L~i}fa"~Y~-'1Y_Si'!•r?$~'t•~r~°a.-.'~in ,.. . _. _ . This model (Figure 8) could allow more complex possibilities, too, particularly if A or.D are in'the mainstream of-primary metabolism.~;Recall the tetracycline,. . ch- case, in;~whiacetate and malonyl coenzyme A are primary metabolites, and sub- .:,.... _ - :jected to many, many controls already pretty well understood. But yoursee the lnt I hope .r.? V' ` ,tiI # Y-r~ . ,urs~ s4 ~~ ;~~~a~ ~PI1 that' eq redis~the determination to investseveral yeain the study of - ri., . .. , . . . , . ....~. ~_~. . i}. :.... . . ._. . _ .. , _.. _. . ,. v°.these mechanisms in the organism of your choice and all this will be possible . . . . s a.W ~- ;» -~ .. . - ..• - _ -. Y'~'4 -~ ~r u+ f;+3:. .~ 1` G 'i ~i~!i+~'s ?«t ~i.~s. :~•J-/if'id'+ rr{ ~... . ; i.~-±n y ~ Another point is worth mentioning.These processes, natural recombination and . . `-DNA stitching,-just-like,mutation, happen in the desired way only in a tiny frac- n.~ -:.tion of theYcells, perhaps one in a millio As with mutation, then,.in the ab- ° -' t;r~`f- sence-o'•a.Darwinian,selection situation, large-scale_cloning and screening will `=probably contiriue to have great relevance.. So at our company, Cetus Corporation, ~;t=, r. we intend to~-combine our large scale screening capability with recombinational =~4 .. . ,,. .. _ .. - _... . . . we have done with mutational programs. and DNA stitching work as ,..... _.,..,. .... - _ , . Finally, I wish to turn our attention to a couple of other topics relating to ~:DNA stitchinFirst there is a development which I believe may make anti ~:' k~3-,., f,<. ":biotics relatively--less`"important within our lifetimes- -it will,.I think, be ~,a ., ,+ •,.~ . _ .~, . ..:,. - . ~~,.--._. ., ..._ •. . ...:,. _ ... pY. • .._1:... - ~...greatly facilitated by' , DNA,stitching I'11 come back to that in a moment. The 3.;other topic is the public concern which has focused on possible hazards of DNA ' everyone feels it's A stitching.:.Since. only a matter of time before facility in . .. ~a;s~ ~ .. -.. moving genes around will,reach the point where even human DNA will find its way , --: yn .th _ . intobacteria, ere-is+worrythatmaybe~~a,.diabolical organism.will.be-created ..~ r and get out of the lab. Maybe it will be a pathogen resistant to all known anti- r =. , ,biotics. -Maybe it will be a bacterium common to the human gut, but inadvertently- made to carry information to make too much of a human hormone or even carrying a human cancer virus. 'These and other nightmares were first highlighted by the y workers in the field themselves.- This was an unprecedented and laudable action. As a resuLt.of their deliberations, we will soon see the promulgation by NIH of guidelines spelling out what are permissible and non-permissible experiments and facilities.* But it isn't clear if that will have any legal clout over ~ institutions, companies, or individuals not working under NIH grants. It cer- ~ " tainly won't apply to work outside the USA. While many workers feel that the earliest fears were exaggerated, we feel that a problem still exists, and that these guidelines should be universally embraced. It is our company's policy to These remarks preceded the.promulgation of NIH Guidelines.

e at least that stringent, and I call on others here and abroad to make similar YpubLic undertakings.**-'An interesting sidelight, known to most of you,r is the ~; Itidevelopment `of research organisms so debilitated in so many ways; that, it- is virtually impossible for them to live"outside the laboratory, or to be more strin- gent, to make it impossible for them to live in the human body. The task of '? r . ~,!creating such "basket case" bacteria has already begun, 'by Curtissl s, andothers y,. ;~This kind of work goes hand-in-hand with the kind of programs I mentioned ~ar-~ r; ~ rg lier to t to roduce ideal industrial roduction microor anisms y f f p ~ Y ,~, ~e ; c . p y~r y~.~tK~I'1i„~ ,'~i+~9Y~s,f~e_~M ~ ; „ ,3 ' We can grow,'{mutate, -wid selectively screen large quantities of industrial micro- . .... . . _ : .. . . . ~, ~.. organisms But for now,"all they praduce is their own native`spectrum of pro-' ~` . . '. ..:r . . . . ,...f - r:. 'ducts -'No process of mutation; or for that matter, recombination among iaicro- _organisms,"will ever cause a aiicroorganism to manufacture insulin,''=human growth or`ari antibody to save a patient dying of encephalitis.~`The changes hormone, in DNA necessary to do that are so complicated that it's statist'ically-valid to say that they will never happen randomly. But it will be possible, some dayfi ~ o stitch into the DNA of industrial microorganisms the human genes to render the microbes'capable of producing vast quantities of vitally needed human proteins.`* z The need for them is great, but until now there was no way to'imagine manufac- ,• .a ~ r 1 G.'>: ~r r3~Y 1 ~fl F u: ,a ~~-l,r) turing them~ ~12- Long before modern science entered the picture, the human body had evolved mech anisms for fighting infection. "(In fact, without these defenses; aian could not have evolved and survived.).~Two major defense systems involve the synthesis of proteins whose function is only now becoming understood at the molecular level. One involves the production of an`anti-viral ccmpound called interferon.- The ; "'`other concerns the much more'familiar protection conferred by antibodies. `j :`d~ a v i.k ~q fw 1 ~ ~~ e,'. In each casthe body produces a'particular protein in very specific'response~ a +to an infecting organism. If the body's defenses cannot respond rapidly enough, ;~ the race is lost.'Before the introduction of sulfa drugs and antibiotics, that meant losing the patient. Yet these life saving antibiotics are`crude indeed in their mode of action ` ~ y ,...a . . ~.:.. . .. ~ .. ...'... ...Y"_,. e specificity of interferon and antibodies is far more exquisite than any ug'ever discovered And they are natural human proteins As such'they may be free of the toxic side~effects characteristic of "drugs " .. . ::~... ,.• .~ <~.~ _ ._.. i..~~ • _:~~ ....'. ~ - ~ :- . But paradoxically,`these ubiquitous proteins; antibodies, and interferon, present : • ' -in every one of us,are virtually unavailable as products to be administered instead of drugs in the practice of inedicine v `iThey are too coinplex to be synthesized chemicallY,• ~ and there is no practical -~~ k_~ ; way to harvest the minute amounts made in our cells.' Furthermore, substantial quantities of these proteins are made only after infection " . . ,. ..... .. ._. .~ . .. . _• Y t ~ ait .t.l_. _ ~ . .. This speech was the first such public pledge by a representative of industry. We currently urge federal legislation to cover all work in this field. :.li~:_ \ , :J.~~ .- . ._ . .. _..... ... .... .•.., . ~L . r , . .... .. ._ . ._ .. .... S .~ ... "This prediction was borne out by recent reports from the University of .'T ** California at San'Francisco.

,:`-Yet, if only a way were found to manufacture these compounds, the practice of. ''iaedicine might be revolutionized. And as~I just said, we believe that it wil : be possible, within the foreseeable future; to stitch, into the DNA of indus-*' ',-\~• trial microorganisms, the human genes to render the microorganisms capable of , 1_producing vast quantities of vitally needed human proteins. 77- . eJr~ The prophylaxis and treatment of disease bywadministration of effective dories := antibodies and.interferon would represent a market which could take its place "" beside, if not largely supplant today's multibillion-dollar antibiotics market.': 'ah , 7 hla.,U«.U was;~'Microbial Genetics and the Future of the Pharmaceutical Industry.".~ I guess my conclusion is that, left to its own'devices, microbial genetics will ~first,.if_,given half.a chance, raise the antibiotic industry to a new high of'; elegance and,efficiency. But then, sometime in•the.-future, microbial genetics . will spawn its own progeny, industrial fermentation of human proteins, and some of these will_compete with antibiotics in the treatment of human diseases. `Which- ever mode of therapy wins that Darwinian competition, it is Man who will end up " most fit as a result ~,~ ~..T 6~~~'~~`«t a~ Vl V P 'x.)~.+')jzfc"S.~`. . .. ~ _.J.. .Trw~~~~. Y-r-.. _ ~.... . . . . ~~_ ' ,~^y.•4..r. - . ,. . . , .. , ;. •.. . . ~ . . _ _ . ' -• . _ .. . _ ?'•. . i ~ i ...,.. ..~G~ ~.~ <: . t6;v i ~ :, . ~ . rv< S ..060 rO ,:1 i.T.iL;US ..-,- ~ -A~~c.~....~ ._.. T. ' ~.__.. .. . _ .._. _._. .. . .•-• . - ~.... t .''7"'.(,I f, . .. . L; -... ~ . "a - - +../ i.f1. ..i :... -S. i .... ... _ ... •.a. .l~ ~.MF.[V.f~.J.A+y•t. ~ r Y Y.: vn~ ~ V'! f:~]~faT~'~'.P~M :......,.. :~. .. ..y'... ..w~ ... ., . . .

Y ( F 13. 1 6 Pontecorvo, G. York. t Hopwood, D.A., 1974. The impact of Genetics on the Study of Antibiotic- Producing Actinomycetes Pt Hi ,osg. I Med. Dosw,, 28:427-439. Jaco b, F., and J. Monod, 1961. Genetic Regulatory Mechanisms of Proteins, J. Mol. Biol., 3 318. Ames, B.N, and P.E. HartTnan, 1963. Histidine Operon. on ant. Biol., 28:349-356. Yanofsky, C., 1964. Gene-Enz yme Relationships In The Bacteria, I.C. G .salas and R.Y. Stanier, eds.; Academic Press, New York, 5:373-417. m 0 ® in.the Synthesis ... _ _. .! ~, Pontecorvo, G., 1976. Presidential Address at the Second International yd~¢ ~ Symposium on the Genetics of Industrial Microorganisms, K.D. MacDonald ed, Academic Press, London, 1-4. >>tw ~ Elander, R.P., 1967 In Induced Mutations and Their Utilization, H. Stubbe, ed., Abh. Deut Akad Wiss Berlin 403423 ...,,-. : . _ . . , z 1 Cohen, S.N., 1975. The Manipulation of Genes. Sci. Am., 233:24-33. 4. Itakura, K., D. Hirose, R. Crea, A. Riggs., H.L. Heyneker, F Bolivar HW ,.. Boyer, 1977 Expression in Elif Ch .. co o aemically-Synthesized Gene for N the Hormone Somatosdadin, Science, 198:1056-1063. . 0 ~; . 15 Curtiss R Apri1 12 1977 Lt .,.,,.eter to Dr. Donald Fredrickson, Director, National Institutes of Health Bethesd Mld 2001 ,a,aryan4.