BUILDING A GENETIC TOOL-KIT
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779 Heckmatt’ suggests that cooperative studies may be needed to assess the value of different forms of treatment in controlled therapeutic trials. Lorbers suggests that the management of neonatal meningitis should not be attempted in any but special centres. Both approaches could yield dividends. However, even with prompt diag- nosis and the best treatment, the scope for therapy may be limited because of the vulnerability of the newborn central nervous system when infected. What of prevention? Nursery outbreaks6 seem un- common, no doubt because standards of nursery hygiene are generally high. Such standards need to be main- tained. The risk of neonatal meningitis is increased in low-birth-weight infants (almost a third of Heckmatt’s series), in infants born after prolonged rupture of the membranes, and in those with other obstetric complica- tions. Cause and effect are hard to demonstrate in these circumstances. However, hopes for reducing the inci- dence of neonatal meningitis may rest, in part, with the obstetric aim of uncomplicated, full-term delivery. BUILDING A GENETIC TOOL-KIT ARMAGEDDON and the cure of cancer shared the same headline when it was announced that a gene synthesised entirely in vitro could function in a living organism. In- terest was doubtless augmented by the near-simul- taneous publication of a recommended code of practice for experiments on "genetic engineering" in the U.K., devised by the working-party under Sir Robert Wil- liams.9 Some of the wilder flights of journalistic fancy might, however, be attributed to the fact that the stories were written on the basis of a Press release, issued as an appetiser before the paper in question was presented. What Prof. Har Ghobind Khorana, of the Massachu- setts Institute of Technology, revealed to his audience at the annual meeting of the American Chemical Society in San Francisco, some days after the headlines had appeared, was in fact a logical development of work on gene structure which has occupied a large team of scien- tists (indeed several large teams of scientists throughout the world) for more than ten years. When the first struc- tural gene was synthesised in vitro,’Othis success was seen to be only partial since functioning genes comprise more than the string of bases, in appropriate sequence of triplets, required to code for the corresponding aminoacids. To take the simplest possible case, that of a gene coding for an R.N.A. molecule, rather than a pro- tein, as the final product; the D.N.A. sequence always contains some bases in addition to those directly comple- menting its R.N.A. transcript. These extra bases include promotor and termination sequences concerned with starting and stopping R.N.A. transcription at the appro- pnate points. Where the initial gene product is a transfer R.N.A. (tR.N.A.) it is usually transcribed in the form of a precursor molecule which must be processed bB a series of enzymes before it is biologically active. 5. Lorber, J Prescribers’ J. 1976, 16, 82. 6. McCracken, G. H., Shinefield, H. R. Am. J. Dis. Child. 1966, 112, 33. 7. Cabrera, H. A., Davis, G. H. ibid. 1961, 101, 287. 8. Szekely, M Nature, 1976, 263, 277. 9. Report of the Working Party on the Practice of Genetic Manipulation (Cmnd 6600). H.M. Stationery Office, 1976. 10. Agarwal, K. L., Büchi, H., Caruthers, M. H., et al. Nature, 1970, 227, 27. The bases coding for all the surplus components of the precursor are, of course, included in the corresponding gene along with the promotor and terminator regions. If the aminoacid sequence of a polypeptide chain, or the base sequence of an R.N.A. molecule, are known, it is a simple matter to write out the base sequence of the D.N.A. for the corresponding structural gene. Even sup- posing we could bridge the yawning gulf between writ- ing out a D.N.A. base sequence and actually synthesising it, such a "gene", lacking the additional non-structural information, would not be expected to function in vivo. What Khorana and his team have achieved in recent years is the addition of the essential promotor and stop sequences to the structural gene for a tR.N.A. precursor molecule from Escherichia coli, building the complete unit of 199 bases (73 non-structural) from short seg- ments of single-stranded D.N.A. They have spliced the synthetic gene to bacteriophage D.N.A. which can then gain access to E.coli cells. This is demonstrated by test- ing the sensitivity of the organism to a T4 phage carry- ing a mutation ("amber") which prevents the utilisation of normal tyrosine tR.N.A. The new gene, coding for a variant suppressor tyrosine tR.N.A., can be used by the amber mutant. This leads to the production of normal phage proteins and hence to lysis of the bacteria. Khorana’s "brick-by-brick" technique of gene assem- bly has two inherent advantages over the alternative approach-synthesis of D.N.A. on a template of mes- senger R.N.A. by means of reverse transchptase." One is that those essential components of the gene which are not transcribed to mR.N.A. can be included in the build- ing process. The other is that the precise structure of the final D.N.A. molecule will be known and there is little risk of "genetic" impurities in the material. This has consid- erable bearing on the safety of procedures in which it may be used. 12 Unfortunately, only a tiny fraction of all proteins has been subjected to complete aminoacid sequencing and the great majority of genes are bound to be much larger than the one which has just been completely synthesised. The practical difficulties of this approach are therefore immense. The way forward will probably entail a two or three-stage technique for gene synthesis, the unique structural sequence of a given gene being synthesised from an R.N.A. template, possibly amplified and purified by a cloning step,12 and then completed by the splicing- on of synthetic promotor and termination sequences, the base composition of which may be, at least in part, common to many genes.13 Such a programme is not going to be completed this year or next, and, even if it leads eventually to the production of a number of genes cap- able of functioning to the extent of generating a protein end-product in a bacterial cell, its application to the in- vestigation or treatment of human disease will require major new advances in both theory and practice. Engineering apprentices traditionally start their train- ing by making themselves a tool kit with which they will later dismantle, repair, and rebuild complex machinery. Molecular geneticists are still at an early stage of their apprenticeship and, despite all the headlines, their acti- vity to date consists of assembling some primitive though powerful tools. 11. Rougeon, F., Kourilsky, P., Mach, B. Nucleic Acids Res. 1975, 2, 2365. 12. Lancet, 1976, i, 893. 13. Szekely, M. Nature, 1975, 256, 365.
Transcript of BUILDING A GENETIC TOOL-KIT
779
Heckmatt’ suggests that cooperative studies may beneeded to assess the value of different forms of treatmentin controlled therapeutic trials. Lorbers suggests thatthe management of neonatal meningitis should not beattempted in any but special centres. Both approachescould yield dividends. However, even with prompt diag-nosis and the best treatment, the scope for therapy maybe limited because of the vulnerability of the newborncentral nervous system when infected.What of prevention? Nursery outbreaks6 seem un-
common, no doubt because standards of nursery hygieneare generally high. Such standards need to be main-tained. The risk of neonatal meningitis is increased in
low-birth-weight infants (almost a third of Heckmatt’sseries), in infants born after prolonged rupture of themembranes, and in those with other obstetric complica-tions. Cause and effect are hard to demonstrate in thesecircumstances. However, hopes for reducing the inci-dence of neonatal meningitis may rest, in part, with theobstetric aim of uncomplicated, full-term delivery.
BUILDING A GENETIC TOOL-KIT
ARMAGEDDON and the cure of cancer shared the sameheadline when it was announced that a gene synthesisedentirely in vitro could function in a living organism. In-terest was doubtless augmented by the near-simul-taneous publication of a recommended code of practicefor experiments on "genetic engineering" in the U.K.,devised by the working-party under Sir Robert Wil-liams.9 Some of the wilder flights of journalistic fancymight, however, be attributed to the fact that the storieswere written on the basis of a Press release, issued as anappetiser before the paper in question was presented.What Prof. Har Ghobind Khorana, of the Massachu-setts Institute of Technology, revealed to his audience atthe annual meeting of the American Chemical Society inSan Francisco, some days after the headlines had
appeared, was in fact a logical development of work ongene structure which has occupied a large team of scien-tists (indeed several large teams of scientists throughoutthe world) for more than ten years. When the first struc-tural gene was synthesised in vitro,’Othis success wasseen to be only partial since functioning genes comprisemore than the string of bases, in appropriate sequenceof triplets, required to code for the correspondingaminoacids. To take the simplest possible case, that ofa gene coding for an R.N.A. molecule, rather than a pro-tein, as the final product; the D.N.A. sequence alwayscontains some bases in addition to those directly comple-menting its R.N.A. transcript. These extra bases includepromotor and termination sequences concerned with
starting and stopping R.N.A. transcription at the appro-pnate points. Where the initial gene product is a
transfer R.N.A. (tR.N.A.) it is usually transcribed in theform of a precursor molecule which must be processedbB a series of enzymes before it is biologically active.
5. Lorber, J Prescribers’ J. 1976, 16, 82.6. McCracken, G. H., Shinefield, H. R. Am. J. Dis. Child. 1966, 112, 33.
7. Cabrera, H. A., Davis, G. H. ibid. 1961, 101, 287.8. Szekely, M Nature, 1976, 263, 277.9. Report of the Working Party on the Practice of Genetic Manipulation
(Cmnd 6600). H.M. Stationery Office, 1976.10. Agarwal, K. L., Büchi, H., Caruthers, M. H., et al. Nature, 1970, 227, 27.
The bases coding for all the surplus components of theprecursor are, of course, included in the correspondinggene along with the promotor and terminator regions.
If the aminoacid sequence of a polypeptide chain, orthe base sequence of an R.N.A. molecule, are known, itis a simple matter to write out the base sequence of theD.N.A. for the corresponding structural gene. Even sup-posing we could bridge the yawning gulf between writ-ing out a D.N.A. base sequence and actually synthesisingit, such a "gene", lacking the additional non-structuralinformation, would not be expected to function in vivo.What Khorana and his team have achieved in recent
years is the addition of the essential promotor and stopsequences to the structural gene for a tR.N.A. precursormolecule from Escherichia coli, building the completeunit of 199 bases (73 non-structural) from short seg-ments of single-stranded D.N.A. They have spliced thesynthetic gene to bacteriophage D.N.A. which can thengain access to E.coli cells. This is demonstrated by test-ing the sensitivity of the organism to a T4 phage carry-ing a mutation ("amber") which prevents the utilisationof normal tyrosine tR.N.A. The new gene, coding for avariant suppressor tyrosine tR.N.A., can be used by theamber mutant. This leads to the production of normalphage proteins and hence to lysis of the bacteria.
Khorana’s "brick-by-brick" technique of gene assem-bly has two inherent advantages over the alternativeapproach-synthesis of D.N.A. on a template of mes-senger R.N.A. by means of reverse transchptase." One isthat those essential components of the gene which arenot transcribed to mR.N.A. can be included in the build-
ing process. The other is that the precise structure of thefinal D.N.A. molecule will be known and there is little riskof "genetic" impurities in the material. This has consid-erable bearing on the safety of procedures in which it
may be used. 12
Unfortunately, only a tiny fraction of all proteins hasbeen subjected to complete aminoacid sequencing andthe great majority of genes are bound to be much largerthan the one which has just been completely synthesised.The practical difficulties of this approach are thereforeimmense. The way forward will probably entail a two orthree-stage technique for gene synthesis, the uniquestructural sequence of a given gene being synthesisedfrom an R.N.A. template, possibly amplified and purifiedby a cloning step,12 and then completed by the splicing-on of synthetic promotor and termination sequences,the base composition of which may be, at least in part,common to many genes.13 Such a programme is not goingto be completed this year or next, and, even if it leadseventually to the production of a number of genes cap-able of functioning to the extent of generating a proteinend-product in a bacterial cell, its application to the in-vestigation or treatment of human disease will requiremajor new advances in both theory and practice.Engineering apprentices traditionally start their train-ing by making themselves a tool kit with which they willlater dismantle, repair, and rebuild complex machinery.Molecular geneticists are still at an early stage of theirapprenticeship and, despite all the headlines, their acti-vity to date consists of assembling some primitivethough powerful tools.
11. Rougeon, F., Kourilsky, P., Mach, B. Nucleic Acids Res. 1975, 2, 2365.12. Lancet, 1976, i, 893.13. Szekely, M. Nature, 1975, 256, 365.
