Recombinant DNA: Scientific and Social Perspectives

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Recombinant DNA: Scientific and Social Perspectives

Vaughn Vandegrift Murray State University Murray. Kentucky 42071

Recombinant DNA technology involves chemical syn thesis or isolation of one or more genes from an organism followed b y insertion into a piece of DNA of a host organism.. . The recent development of a technology known as recombinant DNA has precipitated an unprecedented dehate concerning the rights of scientists to perform basic research. Federal legislation is expected to Lie enacted this year controlling the use of recomhinant DNA techniques. Many scientists have read or heard about recomhinant DNA, hut only a relative few appreciate its scientific and social implications. This manuscript is designed to inform chemical educators not eneaeed . . a s to (1) .. .. in. or aware of. this technolow the n,lture and met hods used in the technology, ('21the rensons for scientific nnd social roncern, and (3) the attemprs made to assuage concerns involving ~ ~ c o m h i n aDNA n t research. Deoxyrihonucleic acid (DNA) is a complex compound responsihle for the transmission of hereditary characteristics (genes). DNA is a macromolecular polymer of nucleotide units joined together by phosphodiester bridges. Four different nucleotide units are normally found in DNA, each one consisting of a deoxyribose sugar group, a phosphate group, and a purine or pyrimidine hase (Fig. 1). Each purine (adenine, ~~~~

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guanine) or pyrimidine (cytosine, thymine) hase is covalently attached to its deoxyribose sugar group through a glycosidic link involving only one of its nitrogen atoms and the first carbon (designated 1') on the sugar ring. Adjacent bases in a DNA strand are linked bv ~ h o s ~ h o d i e s tbridges er involving (Fig. 2;. the 3'and 5' mrhons on theheox).rihose sugar Polvnurleotide strands of DNA thus contain many linked nucieotide groups with a free 5'deoxyrihose end and a 3'deoxyrihose end. Double stranded (double helical) DNA, the form found in most oreanisms. consists of two ~olvmeric . . strands d D N A running in uppui~trdtrections rantipamllel~. 'l'he strands in douhlr. helical DNA are held adiacent to each uther by hydrogen bonding in\dving complementary bases. Adenine is fiurrnallv found hvdronen-bonded to thymine and cytosine is normall; found hidrogen-bonded to guanine (Fig.

PURINES w

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PYRIMIDINES

SUGAR

PHOSPHATE

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0"-

Figure 1. DNA is a polymer composed of deoxyribonucleotides.Deoxyrlbonucleotides contain one mole each of deoxyribose. phosphate, and a substituted purine (A or G) or pyrimidine (C or T). In the deoxyribonucleotldeunit, the deoxyribese is joined to the purine or pyrimidine by a N-glycosidic bond totheN-9 of me purine a t h e N-1 of me pyrimidine and phmphate is present as an ester an the 3'or 5' hydraxyl of the deoxyribose.

Figure 2. Aqacenl oases n a s h a d of DhA polymer are inked by phOsphodiesta bonds tnvolv ng !he 3' an0 S'hydroxy groups ondeoxyrioose. A snandot DhA possesses directianality since it has a 3' end and 5' end.

Volume 56. Number 2,February 1979 1 77

MADAM VM ADAM

A

LIGASE ENZYME

\L D

3'-C-T-T-A-A-0-5' 5'-G-A-A-T-T-C-3'

Figure 3. Double helical DNA consists of two polydeoxyribanucleotidestrands which run in apposite directions. The regular helical nature of the coiled DNA stands permits hydrogen bonding between bases an adjacent strands only when adenine is paired with thymine or when cytosine is paired with guanine (left). Although hydrogen bonds are weak attractive forces, the large number of them between camplementaly baser in DNA contributes to stabilization of the double helix. The double helical structure is also stabilized by hydrophobic interaction between stacked base pairs along the helix. A gene is a linear sequence of bases along a DNA strand.

Figure 4. The action of a restriction endonuclease. EcoR1. EcaRl hydrolyzes phosphodiester bonds at specific sequences (palindromes)along a double helical DNA molecule. A palindrome contains a twofold rotational axis of symmetry which reads the same frontwardsor backwards from the axis of symmetry. (a) if the DNA has been chemically modifiedat the sites indicated by the asterisks, EcoRl cannot cleave. (b) Unmodified DNA forms recognition sites termed "bubbles" at palindromes by intrastrand hydrogen bonding and is cleaved by the enzyme. (c)Cleavage of unmodified DNA results in the formation of tsils of DNA bases (sticky ends) which may hydrogen bond to complementary bases on any piece of DNA produced by cleavage with EcoR1. (d)Hydrogen bonded Sticky ends may be reformed into an intact double helical DNA molecule using an enzyme (ligase) which reforms phosphodiester bonds.

3). The information known as genes is stored in the sequential arrangement of the four nucleotide bases along a DNA strand. T h e arrangement of bases acts as an encyclopedia which is "read" by the cell in the production of DNA copies composed of ribonucleic acid (RNA) in a process known as transcription. The information transcribed into RNA from DNA is decoded in the synthesis of protein (translation). The genetic code is a triplet code since three sequential bases within a gene code for the introduction of one amino acid residue into protein during translation. Recombinant DNA technology involves chemical synthesis or isolation of one or more genes from an organism followed by insertion of this DNA into a piece of DNA of a host organism (recombined DNA) in such a form that the host organism will copy the inserted DNA. Once reproduced (cloned) in the host organism, the inserted DNA or its gene product (e.g., protein) may he isolated and purified from the host cell. The development of recombinant DNA technolorn .. was dependent upon 11 I informatiw accumulated oter a period of years otl E X ~ ~ r i c h oi ud i bartwin and its genetic characteristics, (2) the discovery of a class of enzymes, known as restriction enzymes, which break phosphodiester bonds of DNA a t specific sites, (3) the development of techniques, using ligase enzymes, to splice together DNA molecules which have been generated by treatment of DNA molecules with restriction endonuclease enzymes, (4) the development of vectors for carrying DNA genes to be cloned, and (5)the development of techniques for inserting and identifying recombined DNA molecules reproduced in a host cell. E. coli is the host organism most widely used for cloning of recombinant DNA molecules simply because more is known about its genetic makeup than about any other cell type. E. coli bacteria are unicellular organisms classified as prokaryotes because their cells do not contain membrane-limited nuclei, as do the cells of higher organisms such as yeast or man. Derivatives of a snecific strain of E. coli K-12 are most often used in recombinant DNA experiments. E. coli bacteria contain a sinale chromosome. c o m ~ o s e dof a circular double helical DNA molecule, which contains genetic information

controlling the growth and development of the cell. In addition to the chromosome, separate extrachromosomal elements known as plasmids are often present which carry, among other things, genes providing resistance for the bacterium against certain antibiotics ( I ) . Plasmids are also circular DNA molecules. but are much smaller than the chromosome and circular~; closed in a manner which makes them easy to isolate and separate from the chromosomal DNA. Thus. plasmids are physic& distinct from the hacterial chromosome and are not normally required for the growth and maintenance of the cell. Certain E. colt strains may be infected by a DNA-containing virus (bacteriophage) such as phage lambda which may propagate itself in the bacterial cell as an extrachromosomal element which is released upon lysis of the hacterial cell (2). Plasmids.. .ohage and " lambda. or combinations of nhaees " plasmids serve as excellent agents for cloning of recombinant DNA molecules. Extrachromosomal elements used to clone recombined DNA are known as vectors. Naturally occurring plasmids and phages have been genetically manipulated by scientists to enhance their usefulness as vectors in cloning genes. DNA originating from any organism, or chemically synthesized DNA, inserted into vectors may be propagated by the host hacterial cell independently of genetic function specified by the chromosomal DNA (3). Nucleases are enzymes which hydrolyze phosphodiester linkaees in DNA either from the ends of the oolvnucleotide cllaini lexmuclenses) or at sites internal along t h t polynuclwtide chain lendonucleases~.Restriction endonurleaws :in. a class of bacterial nucleases which serve the bacterial cell by recognizing foreign DNA (e.g., infecting viral DNA) and destroying it by cleaving phosphodiester bonds a t specific sites depending upon the identity of the enzyme (Fig. 4). The resulting large segments of DNA produced by restriction endonuclease action are then degraded completely by other exonuclease enzymes present in the hacterial cell. Restriction endonucleases thus act aspart of an "immunesystem" for the hacterial cell hy initiating the destruction of foreign DNA thereby restricting its ahility tn propagate. In order to protect

78 1 Journal of ChemicalEducation

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itself from its own immune system, the hacterium has modified its DNA (generally by methytation of nucleotide bases) a t specific sites so that its restriction endonucleases cannot initiate - - ~ the ~ destruction ~ ~ ~ ~ - of its own DNA ( 4 ) . Even newlv renlicated (duplicated) bacterial DNA escapes r e ~ t r i c t i o ~ s i n c e restriction endonucleases will not restrict DNA in which at least one strand of the double helix is modified. In order to brine about successful clonine of recombinant DNA vectors inseFted into a hacterial cell,it is necessary to use a mutant bacterium which cannot restrict the recomhinant DNA ( 5 ) . The most useful restriction endonucleases isolated from hacterial cells and used in recomhinant DNA research are those which produce DNA segments with cohesive (sticky) ends. Sticky ends are regions of complementarity of nucleotide bases whiih result from the fact t h a t restriction enzymes cleave only a t recognition sites known as palindromes (6). ~alindron&arr s&enres of two-fold rotational symmetry. An rxnmple uf an English palindrome is, "MADAM I ' l l ADAM." A nalindnmic seauence in a douhle helical strand of DNA is exemplified by . ~

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CTTAAG GAATTC

through which exists a central axis of symmetry. Presumably, restriction enzymes recognize palindromic sites since they contain regions of intrastrand i ~ m ~ l e m e n t a r iwhich ty may result in a "bubble" formed in the douhle stranded DNA. Treatment of a circular unmodified plasmid, containing only one restriction site, with a specific restriction endonuclease will generate a douhle helical DNA molecule with cohesive ends (7). Through complementary base pairing this piece of DNA may be joined to any segment of DNA which contains the corresponding base pairing complements on its cohesive ends. For example, a DNA molecule containing a gene may he obtained by treatment, using a specific restriction enzyme, of DNA isolated from human cells. The gene can then be ioined to~ n-~hacterial , .-. ~ ~olasmid. which had been treated with the same enzyme, and annealed to form an intact recombinant DNA molecule. A liease enzvme anneals DNA molecules hv catalyzing the formation of phosphodiester bonds a t sites along DNA where bases are properly paired (Fig. 5) (8). Isolation of DNA from cells, followed by treatment with restriction enzymes, is only one method for obtaining DNA molecules for recombinant DNA experiments. Once the amino acid seauence of a neotide or protein is known, a sequence of DNA which may code for that peptide or protein can be predicted and synthesized chemically. DNA may also be synthesized from isolated RNA molecules by a process known as reverse transcription. However, DNA molecules synthesized chemically or by reverse transcription may not contain restriction sequences (palindromes) as part of their structures. The addition of a soecific seauence to the end of the DNA molecule allows fo; insertion of the DNA molecule into a bacterial nlasmid or other vector at the desired site, usinn the technique' outlined in Figure 5. Recently a butt l i g i e enzyme, which does not reauire stickv ends to catalyze the annealing of DNA, has been isolated (9) and used to anneal restriction seauences to DNA molecules. Simply being able to splice two or more fragments of DNA together is not enough for successful recomhinant DNA experiments. The recombined DNA must be of such a nature that it can be copied in the bacterium, preferably in multiple copies. Some very small plasmids carry genetic information which allows them to achieve 100 or more copies in the cell (10).Amnlification of these small olasmids is not normallv deliteriois to the growth of the cell,since growth is controlld by the genes present on the chromosomal DNA. Vectors such as plasmids or viruses containing recombined DNA spliced together as described above may he taken up by the bacterial cell when placed with the cell in a dilute solution of calcium chloride. Once the cell has taken up the plasmid amplification occurs, unless the inserted DNA is harmful to the cell, and any genetic markers characteristic of the plasmid (e.g., antibiotic ~~

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resistanre) become chararteristic of the host cell which is said to have heen transformed. Bv using vertors containing antibiotic resistanre it is possihle not only to determine whirh horteria have heen transformed by the recombinant DNA, hut also to grow colonies of the transformed bacteria since they will now he resistant t o certain antibiotics (11). Althoueh recombiued DNA molecules mav oroduce multiple copies of the DNA in the bacterial cell, &e genetic information is required for the synthesis of a protein from the cloned gene. The genetic control mechanisms of a eukaryotic cell (higher-order organism) are sufficiently different from a prokaryotic cell (hacterial cell) that insertion of a eukaryotic DNA fragment on aplasmid which may be capable of coding for synthesis of a protein does not guarantee that the protein will he synthesized. In order to ensure that protein synthesis from the cloned gene will he effected in the bacterial cell, the cell's protein synthesizing system must he tricked into recognizing the foreign gene as part of its DNA. Such trickery can he accomplished by carefully splicing the foreign DNA in the vector next to some control elements (gene switches) characteristic of the hacterium. Somatostatin is a mammalian pentide hormone composed of 14 amino acids normally produ.ced in the hypothalamus at the IIRSC of the brain. Somatostatin inhibits the secretion of a number of hormones, including growth hormone, insulin, and glucagon. The effect of somatostatin on the secretion of these hormones has attracted attention t o its potential therapeutic value in acute pancreatitis, and insulin-dependent diabetes. Somatostatin, along with several other hormones, was initially isolated in milligram quantities by extraction of ground-up brain tissue of a half million sheep. To demonstrate the utility of the recombinant DNA technique, researchers recently brought about the synthesis of somatostatin in a hacterial cell (12). The DNA for the somatostatin gene was chemically synthesized from nucleotide precursors rather than isolated from mammalian DNA by restriction endonucleases. Using chemical techniques, units containing nucleotide portions of the gene were synthesized. Ligase enzymes were then used to link the gene portions together, producing the complete douhle helical DNA molecule

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Figure 5. Outline of a recombinant DNA experiment using a bacterial plasmid containing one EcaRl cleavage site. Isolated bacterial piasmids are subjected to E m R l cleavage. generating sticky ends which can hydrogen bond to a segmem of an exogenous DNA molewle containing lhe wmplementaly sticky ends. Once the comolementarv base oairs have formed. the inserted DNA is made a oart ot the bactwial ol&mid usino the lioase enrvme. Bacterial cells are lhen exposed to tne plasmla mder condrt ons res.lt ng m the ~ p t a k eof the P asma ttranslormalmn, Theenem of the procedure 0s lnst OhA segments to oe c onw are inserted into the bacterial cell.

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Volume 56, Number 2 February 1979 1 79

Figure 6. Outline of an experiment used to bring abovt expression In E. colt of a chemEcaity synttmired gene f w the mammalian hamone somatostatin ( 12). Chemically synthesized DNA was inserted into a bacterial plasmld by an eiabm t e prDcedure based cm lhe techniques outlined in Figures 3 and 4. The inserted DNA was olaced in the middle of a DNA seament on the ~lasmidccdina tor a oactcr a1 proler m a next lo a bacler al gene sw lch to e n w e exprersoon of the clonea DNA The recomb~nantplavm.d was wed to transfwm E cohand a protein product was produced in vivo which consisted of a bacterial protein containingsomatostatinas a portion of i h structure. The somatostatinpeptide was removed from the bacterial protein carrier by chemical cleavage.

gene therapy, and immunotherapy against viral disorders and cancer. For examule. in immunotherauv. that Dart of theviral genetic material.which codes for syithesis df the viral coat urotein could be isolated and cloned in E. coli. The urotein produced could then serve as an antigen to elicit the immune response in an individual, thereby avoiding the use of potentially dangerous intact attenuated or inactivated viruses in vaccines (14). Current experiments involving recombinant DNA offer a means of placing genes of one organism into non-chromosomal elements of bacteria, producing vectors capable of transforming susceptible host cells to copy or express genes present on the vector. The host oreanism does not take on anv char" acteristics of the organism from which the foreign genes were isolated other than the presence of small pieces of DNA or synthesized protein. The controversy concerning the use of recombinant DNA is based on sneculation as to the suitabilitv of the host organism (E.coli) and its vectors, and on what may or may not occur when certain gene recombinants are used to transform E. coli hosts. As early as 1971, as the ability to generate recombinant DNA molecules developed, scientists voiced concerns about possible hazards of the research (15). Scientists had known for years that recombination of DNA occurs naturally since genetic traits often appear in progeny which are not found toeether in either of the Darents. However. it was eenerallv ac&pted that exchange of genetic iniormiition was &cludeb hetween orranisms of different sr)eries.The ahilits to comhine genetic information from v e j different organisms placed scientists in an unknown area of biology. I t is not sururising, therefore, that placing human genes bacteria seemed t i o risky to some scientists. Private concerns crystallized in June 1973, a t the Gordon Conference on nucleic acids, when some 90 scientists decided to publish a letter of concern in Science, and to request that the National Academy of Science (NAS) appraise the risks (16). The committee, formed by the NAS, met in April 1974,and recommended a temporary moratorium on certain types of recombination research (17). In addition, the committee called for an international conference on recombinant DNA. The international conference convened a t Asilomar, California, in February 1975. The participants agreed that most of the work on construction of recombinant DNA molecules should proceed provided that appropriate safeguards adequate to contain newly created organisms were employed. The moratorium on "hieh risk" e x ~ ~ r i m e nwas t s continued and w,as asked toestilblish the National ~nstiru't'rso i ~ e a l t ' h(XIHI ruidelinei. One ut the most sienifiranr outcomesof the Assiyomar conference was the ideathat biological containment of host-vector svstems could he useful in addition to uhvsical containment safeguards. Biological containment b&rikrs of two types were proposed, including the development of: (1) fastidious bacterial hosts unable to survive in natural environments, and (2) equally fastidious vectors (plasmids, bacteriophages) able to grow only in specified hosts and unable to move from the specified hosts to some other host (nontransmissible). Physical containment, exemplified by the use of portable hoods or negative pressure laboratories, would provide an additional factor of safety when coupled with biological containment (18). Bioloeical containment. it was reasoned. would reduce fears s h u t placing fdreign gene.; in E. ro11 ns well as reduce fears about the use of E. coli itself. The natural habitat oft'. colt is the alimentary trart of man and many warm-hlonded animals. E c d i is also rarried by many other organisms, such as fish and insects, and rim be found throughout the biosphere in soil nnd water. Ihder normal conditions E. coli is non-pathogenic. Some strains of E. coli can make substantial cont;ibuti&s to human disease, especially urinary tract infections, post-operative wound infections, and diarrhea. The particular strain of E. coli, designated E. coli K-12, which has been used for many recombinant DNA experiments is a laboratory strain

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containing the somatostatin gene. The somatostatin eene was synthesized with specific cohesive ends so that it could be recombined with a vector plasmid which had been treated with a suecific restriction enzvme. After lieation the recombined DNA was used to transiorm bacteria. The presence of protein synthesis control regions on the plasmid adjacent to the inserted somatostatin DNA allowed for synthesis of the rotei in. In earlv exueriments somatostatin could not be detected since the proteolytic (protein-degrading) enzymes of the bacterial cell recoanized the somatostatin neutide nroduced as being foreign. T o circumvent this probl& the somatostatin DNA was incorporated a t the end of a bacterial DNA segment coding for a normal bacterial protein. The 14 amino acids comprising somatostatin were present at the carboxy-terminal end of the synthesized protein. The presence of a large piece of bacterial protein was sufficient to prevent the somatostatin ueutide . . from beine deeraded and it also prevented somatostatin from being an active hormone upon isolation. T o recover active somatostatin from the attached carrier protein, the protein was treated with a chemical which released the free somatostatin peptide. The peptide bond a t a methionine residue, which had been inserted immediately preceding the first amino acid in the somatostatin peptide, was cleaved using chemical methods involving cyanogen bromide (Fig. 6). The somatostatin experiments underscore the potential of recombinant DNA technology. About 100 grams of bacteria was erown in auoroximatelv two eallons of culture. resultine .. in the "manufacture" of milligram quantities of somatostatin, thus eliminatine the reauirement for laree amounts of mammalian brain tissue. certain other hormones, enzymes, blood factors. and exuensive uhmaceutical ~roductsmav somedav be producrd b; recom.hinnnt DNA techniqurs. ~esearrher's at the llniversitv of California at San Fnmcisco predict that by 1982, E. coliwill be used to mass-produce hsulin (13). Currentlv, most recombinant DNA ex~erimentsare being performed as a tool to study the organization and structure of genes in eukaryotic cells. Manipulations of genes a t the recombinant DNA level may help to unravel problems ranging from cancer t o increased agricultural crop productivity. Other applications of recombinant DNA are foreseen in

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80 1 Journal of Chemical Education

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Brlet ovtllne of t h e guidelines for recombinant D N A research promulgated b y L e National I n e i t u t e of Health in 1976.

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Four levels of physical containment aredesignated, P I toP4, with P1 being the least stringent and P4 the most. Physical containment levels are combined with W e biological containmem levels. EKI to EK3, f w each recombinant DNA experiment according to potential risk. The least dangerous experiments are classified P I EKt a n d w mostdangemusexperimentsP4 EK3. Same levels specified for recombinant DNA sources far shotgun experiments using E coli as the hast and plasmids, bacteriophagesw o w r viruses as the cloning vectws are:

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Embryonic primate tissue or germ line cells Cold blwded vertebrate germ line cells Cold blooded vertebrates which produce a toxin Prokawote~which exchange - high risk pathogens with E. Cali Plant viruses

+ + +

P3 EK2 P2 EKl P3 EK2 Banned

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Animal Viruses Prokeryde~which exchange nonpathogenic genes with E. Coii Birds Mammalian cells Animal viruses which may contain harmful genes Plant which do not contain pathogens

+

P3 EKl or P2 EK2 P3 EK3 or P4 EK2 P1 EK1

+ + + +

+ EKI EK2 + EK2 + EKl

P3 P3 f P4 P2

*AM m e g u i ~ i n e s m ebiding only oomose rewcherr MWw i governmentgrants. the entire scientificcommunity has agreed to comply. The passagage of tederai legislation basedon the ~ i ~ g u i d e i i n 1 essimminent.

u hich was isolated 50 yenrs ago from a human patient. There are sionif'irant d~fferencesbetween K-12 and the maiorits . . of E. co; strains. For example, E. coli K-12 does not normally colonize the human intestine even when 10" organisms are ingested, hut it may do so under unusual conditions such as during antibiotic therapy. Even when it cannot colonize, a small fraction (10-8) of E. coli can still pass through the human intestine unharmed (19). As NIH was developing guidelines for recombinant DNA research, Roy Curtiss of the University of Alabama at Birmineham. was husv develo~inea safer host strain of E. coli K-1; niededto do chat resenrchQ0). The development of the strain was morc difficult than anticioated takine I8 months to complete. In March 1976, the ~ u i t i s sderivacve of K-12, dubbed Chi 1776,was certified as meeting NIH guidelines for a safer host (21): Chi 1776 and subsequent derivatives of it cannot survive outside of a laboratory. Among other things, these strains require certain laboratory chemicals, are sensitive to antibiotics and bile salts, and are destroyed by sunlight. In addition to the efforts of Curtiss, other scientists were developing non-transmissible vectors to be used in recombinant DNA research (22). The NIH released recombinant DNA guidelines in June 1976. The guidelines explicitly prohibited certain types of experiments and suggested four levels of physical containment and three levels of biological containment for use in recombinant DNA experiments. Experiments where there was the most uncertainty of the risk involved, such as shotgun experiments, including the insertion of random DNA segments of primate DNA, were classified a t high levels of physical and biological containment (P4 + EK2 or P3 EK3) (Fig. 6). The release of the guidelines did not end the debate. Scientists on lrnh iidcs of the issue, arguing in thr p~tl)licarma. attrncted much stcention and s~lpportfor their views. National attention fncust~Ion the debate in the summer of 1976 when t hr city of Camhridge, Massschusettr discuised the intcntim of' Harwrd L'niversitv to 1)uilil n moderate risk (1'31 recombinant DNA laboratory in the city. In contrast t o the NIH committee. which was comoosed mostlv of scientists. the committee appointed in Carrhridge was cbmposed of layken. Much debate ensued, hut when the city ordinance controlling recombinant DNA research was finally passed, it was only slightly more restrictive than the NIH guidelines. During recent congressional sessions several hills have heen introduced concerning recdmhinant DNA but none has been

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passed. One reason for this is that new discoveries are constantly influencing congressmen. Some scientists have presented their latest exoerimental results in Washineton before they were published i;l the scientific literature to inhence the nature of the leeislation. The most recent examoles are the somutostatin w;;rk discussed above and the woik of Shing Chane and Stan Cohen of Stanford I.'niversitv, which was puhliihed in the scientific literature in No&nber 1977 (23). The work of Chang and Coben attempted t o resolve an armment of those scientists opposed to recombinant DNA experiments which centered o n t h e concept that barriers in nature exist against gene transfer between bacteria and higher-order organisms. They argue that to break this barrier in the laboratory is to tinker with millions of years of evolution. Certainlv. develonine gene combinations which nature strictlj, forbids'could potentially release to the environment recombined molecules with hazardous conseauences. The work of Chang and Cohen diminished the forcefulness of this argument. Their findings indicated that fragments of eukaryotic DNA, although unmodified (unmethylated), could he taken up into a bacterial cell, escape destruction, and he ligated into bacterial plasmid DNA (24). Although the probability of such an occurrence is low, the fact that recombined DNA plasmids were produced in viuo suggested that recombined DNA molecules involving genes from man may have already been produced from interaction between bacteria and human DNA in the intestinal tract. Knowledge that such an occurrence is oossihle removes some fear about how daneerous the generatio; of recomhinant DNA molecules is. ~ a t h & than being forbidden in nature, recombination of DNA from different organisms may occur and, as such, may constitute a mechanism of, rather than a harrier to,evolution. Although the work of Chang and Cohen has influenced the thinking of some scientists and congressmen, others argue that the experiments were performed under unusual laboratory conditions not duplicated in nature. Annthrr rEcent development in the drbate is the discovery that se\.eral, and perhaps many, of the genes in eukaryotes contain segments-within the DNA sequence of most genes which are not translated into protein (25). As discussed earlier, certain RNA molecules act as intermediates between DNA and the synthesis of protein by carrying the message from the DNA eene to the site of protein svnthesis. Eukarvotes presumably contain systems capable of cutting up and splicing together portions of the RNA molecule produced from the DNA gene. The difficulty observed in cloning eukaryotic proteins in E. coli in early recomhinant DNA experiments h a y reflect the fact that bacteria lack the cutting &d splicing system of eukaryotes. The synthesis of certain eukaryotic Goteins mav therefore he deoendent on the develonment of suitable eukaryotic host vector systems. I t is interesting to speculate that the cutting and splicing system which bacterial cells lack may constitute the genetic barrier which has been proposed to exist between prokaryotes and eukaryotes. The development of safer host organisms and vectors for clonine recombinant DNA molecules coupled with early recom1;inant I)NA experimental rtwlts have shifted thk emuhasis of thr drbare. Questions now cnncern the cmditims under which the research will he performed rather than whether or not most recombinant DNA research should he prrfmmrd. Hisk assessment experiments rurrentlv underway at Fort 1)etrick. Maryland, should help to establish the risks invdvrd in the nwarch. Meaou,hile. the NIH euidelines. although only binding on those investigators hGding all government erants. are heine followed voluntarilv bv all researchersin the'united states. Indications are that the scientific community will he allowed to continue most forms of this research under safeguards similar to the NIH guidelines which will be specified in federal law. A

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Acknowledgment The assistance in the preparation of this manuscript of the Volume 56. Number 2, February 1979 1 81

following students enrolled in a Special Topics in Biochemistry course a t Murray State University is thankfully acknowledged: R. T. Allen, D. G. Davis, R. R. Evans, S. R. Hamaan, P. G. Hunt, S. M. Johnson, T.C. Johnston, E. G. McFarland, R. E. Oliver, T. H. Pritchett, S. K. Rhodes, S. D. Stayton, and D' A' Stewart' The author was a participant in an NSF Chautauqua-Type Short Course on Recombinant DNA, and would like to thank the National Science Foundation, the American Association for the Advancement of Science, and Drs, Betty Kutter and Leroy Walters for their support of that program. Literature Cited (I! Adams,RL P.,Bumon,R.H.,Cmpbell,A.M,andSmeUic.RM.S.."Dandmn'sne Biochomiatry ofthe Nueleie Acids," 6th Ed., A a d . Press. New York, 1976 p. 150. (21 Davis. B. D.. Dulkaeo. R., Eisen, H.N., Ginsberg. H.S.. and W d . W. B., "Microhiolagy," 2nd Ed.. Harper and Raw. New York. 1973, p. 1091. 13) Sinsheimer, R. L.. "Annual RavicwofBiochemDtry~Annual Reviem, 1nc.PaloAlto. California, 1977.46. p 418. (4) Kornherg. A.,"DNA Synthesis," W. H. b e m a n & Co., San Fraoeko, 1974, p. 315.

82 1 Journal of Chemical Education

(5) Ra'(3hp.418. (61 Natham, D., and Smith. H. 0,"Aonual Review ofBioehemiatry:Annual Ine., Palo Alta, California, 1975, p. 275. (7) C o h e n , ~~. . . S c i e n . ~ m a r26 . (JUIY19751. ~ ~ ~ ~ b ~ s ~ $ . Noll. , R o c .

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