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Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009). Vo133. No.8, August 1987 CENTRAL AFRICAN JOURNAL OF MEDICINE 9. EvansJS, WennbergJE, McNeil BJ. The influence of diagnostic radiography on the incidence of breast cancer and leukaemia. N Engl J Med 1986; 315: 810-15. 10. Philpott RH, Castle WM. Cervicographs in the management of labour in primigravidae. J Obstet Gynaecol Br Commonw 1972; 79: 592-8. 11. Armon PI The management of patients previously delivered by caesarean section. Cent Afr J Med 1971; 17: 170.-2. 12. Walton SM. The antenatal and intrapartum management of patients with previous caesarean section scars. East Afr MedJ 1978; 55: 1~8. 13. Singh TKC, Barman SD. Gupta AN. Study of vaginal delivery in patients with one previous lower segment caesarian section. Aust NZ J Obstet Gynaecol 1986; 26: 245. 14. Demianczuk NN, Hunter DJS, Wayne Taylor D. Trial of labour after previous caesarean section: Prognostic indicators of outcome. Am J Obstet Gynaecol 1982; 142: 640-2. 15. Jagani N. Schulman H, Chandra p. Gonzolez R, Fleischer A. The predictabilily of labour outcome from a comparison of birth weight and X-ray pe1vimeuy. AmI Obstet Gynaecol 1981; 139: 507-11. . SPECIAL ARTICLE already apparent in such developments as recombinant human insulin, prenatal diagnosis of genetic disorders, and the studies of relatedness among various strains of Human Immunodeficiency Virus (HIV). In the near future we can expect to see applications of this technology having an impact on many more areas of medicine in Zimbabwe as well as elsewhere. The basic 'double helix.' structure of DNA as described in 1953 by Watson and CriCK is illustrated below to refresh ~our memory (Fig. 1). The DNA is transcribed to messenger RNA which in tum is translated into proteins according to a code which is consistent in all life· forms. Several developments have contributed to the recent explosion in knowledge and practical applications of DNA molecular biology. These include the use of restriction endonucleases, nucleic acid sequencing techniques, and the ability to manipulate 'vectors' such as plasmids. 'Restriction endonucleases 'are enzymes which have the property of cutting DNA at very specific points. For example, an enzyme called 'Eco RI' m~es a cut only when it encounters the nucleotide sequence GAATIC and the endonuclease Sma I cuts only at CCCGG. These sites provide 'landmarks' in long strands of DNA and enable a type of mapping of an organism's genome (Fig. 2). Advances in DNA Molecular Biology and Their Clinical Applications: Update for Clinicians S HOUSTON SUMMARY Major advances have occurred in understanding and applications of DNA molecular biology. Selected DNA fragments can be 'cloned', allowing manufacture of their protein products. Examples include recombinant insulin, interferon, and vaccines using recombinant antigens. DNA 'probes' are already proving valuable in microbiologic diagnosis and may have an important role in prenatal diagnosis of inherited disease. DISCUSSION So much has happened in the field of molecular biology in the last decade, that it calls for an update on the basic science that we were taught in medical schooL The fruits of this increased understanding are Department o/Clinical Pharmacology, Godfrey Huggins School 0/ Medicine. University a/Zimbabwe. 208 Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009). Vol 33. No.8. Augult 1987 FIGURE I - The DNA molecule, demonstraling the specific pairing of complementary nucleotides and the double helix structure DNA STRUCTURE PROTEIN § METHIONINE <.) I §'" k r <.) fT A C A V rr; C . G C G C T ···A C<o·G G"O 3 5 i ...z A r: GLYCINE 0 T "'A § SEAINE I . § ISOlEVCINE ; u ~ % § GLYCINE .. <.) z ~ ALANINE ~ AlANINE u 3' 0=911"'''''' C=cytosine A=adenineZT=lhymlne U=uracll FIGURE 2 -Restriction endonuclease 'map' ofthe HN genome. Arrows indicale the sites at which virus DNA is Clll by specijic enzymes. The 'pol' gene codesfor viralDNApolymerase (reverse transcriptase) and the 'env' gene for the envelope proteins RESTRICTION ENDONUCLEOSE MAP OF HUMAN IMMUNO-DEFICIENCY VIRUS ~ J. =- 1 ~~ ~ 1 1 1 )(1 r ~~~~~t) 11K if pol=gene encoding reverae transcriptaae env=gene encoding the envelope protein the pattern of fragments produced by a given enzyme wUl be unique to thai. species. Moreover, the fragments are of a manageable size facilitating With a battery of these enzymes the genome of an organism can be cUt into fragments. Since an organism's nucleotide sequence is highly specifIC, 209 Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009). Vol 33. No.8, August 1987 CENTRAL AFRICAN JOURNAL OF MEDICINI! study (e.g. nucleotide sequencing) and manipulations such as insertion into vectors. A vector such as a plasmid is an extrachromosomal bit of DNA ina bacterial host cell (Fig. 3). We -~ f:lmiliar with plasmids as agents with genes ~....uing for antibiotic resistance. A plasmid can be manipulated, for example by inserting a fragment of foreign DNA into it The fragment will then be replicated along with the rest of the plasmid in the bacterial culture. That DNA fragment has now been cloned. IT it incorporates a desirable gene, that gene can now be produced in unlimited quantities in cultures of host cells. Under the right conditions these cells will use their cellular machinery to produce the product encoded by the inserted gene. Thus, we have a mechanism for producing unlimited quantities of: (I) a desired gene or DNA sequence, and (2) consequently, a desired gene product such as a protein. Below are a few examples of the ways in which these techniques can be used. organisms. A recombinant DNA malaria vaccine, using sporozoite antigen produced by Plasmodia genes cloned in E. coli, is undergoing human clinical trials. Hepatitis B vaccine produced in yeast cells is now commercially available. It avoids the (unfounded) concerns about the use of the serum-derived vaccine and may reduce production costs in future. An elegant embellishment on this process is the incorporation of cloned genes for desired antigens into the genome of the vaccinia (smallpox vaccine) virus. This virus then expresses the desired antigen and stimulates specific immunity while offering the potential advantages of a live vaccine. This approach was used in the wellpublicized French HIV vaccine currently undergoing clinical trial in Zaire. DNA technology has advanced other areas of biological research as well. While the sequencing of amino acids in proteins is very difficult. the sequencing of the genes which code for them is relatively easy. This has greatly increased our understanding of' the structure of important biological proteins, including enzymes and the structural proteins of receptor sites. FIGURE 3 A DNA fragment containing a desired gene (shaded) has been inserted into an E. coli plasmid; it has now been 'cloned'. E.COLI CELL Co chromosome Gene mutation Genes which Can be identified and cl~ned can also be intentionally modified structurally. One present use of this capability is in the study of virulence factors and pathogenic properties of micro-organisms. For example, IgA protease, an enzyme which degrades mucosal antibody, had been postulated to be an essential virulence factor in Neisseria gonorrhea. This theory was disproVed by observing the unchanged virulence of strains from which the IgA protease gene had been deleted. In future. the ability to manipulate and alter genes may offer the possibility of curative treatment of genetic disorders. This is a complex subject on its own which raises many ethical as well as scientific questions, and which is beyond the scope of this article. plasmid containing inserted DNA fragment The gene for human insulin has been cloned in bacteria allowing the production of a product which is not only pure, but unlike pork or beef insulin. chemically and anti genically identical to natural human insulin. This product is already in commercial production. Using the same principle, many difficuIt-to-obtain human biologic products such as interferon, growth hormone, erythropoeitin, etc. can be synthesized in unlimited quantities. Genes encoding antigenic proteins can be cloned in the same way and the antigens themselves produced. The resulting product is not contaminated by potentially cross-reacting or toxic materials as is often the case with antigen extracted from whole DNA probes Diagnostic DNA probes are already commercially available for a few organisms but they are likely to assume a far greater role in the near future. Although caution is always appropriate in proposing 210 Vol 33, No.8, August 1987 CENTRAL AFRICAN JQL'Rl"AL OF MEDICINE' 'high tech' solutions in developing countries, some newer probes will probably be quite modest in their technological requirements and cost. Furthermore, they should, at least in some circumstances, prove a satisfactory and relatively cheap alternative to such costly technology as viral culture, The principle underlying the use of DNA probes is fundamentally simple, taking advantage of two basic characteristics of the DNA molecule: (1) The nucleotide sequence of an organism's DNA is highly specific to that species, (2) Single strands of DNA which are complementary bind to each other avidly, following base-pairing rules, to form doublestranded DNA. The probe begins as a fragment of DNA excised from the desired species, cloned in quantity, and then tagged with a radioacti ve or enzyme label. The result is a tagged molecule which will bind only to a complementary strand of DNA, i.e. one from the same species (Fig. 4). DNA or RNA, or both, a probe for any organism bacteria, fungus, virus, rickettsia, mycobacteria, parasite, or even plasmid, can easily be designed, Probes for mycoplasma and legionella have reached the commercial stage. Probes have been developed for Epstein Barr virus, cytomegalovirus, P./alciparum, M. tuberculosis. the plasmid of enterotoxigenic E. coli, and many other organisms. A somewhat similar approach can be used to diagnose abnormalities of the genome in hereditary human disease. Two examples are the antenatal diagnosis of B. thalassemia and, using a somewhat more complex method,ofHuntington' s Chorea. Probes have been used to demonstrate viral DNA integrated into the cellular DNA of certain tumours. For example. the finding of Hepatitis B virus DNA incorporated into the genome of hepatoma cells adds to the evidence that the virus plays an aetiologic role in inducing liver cancer. Under certain laboratory conditions, strands of DNA of related. but not identical. organsims can be made to hybridize. By varying these conditions. an accurate estimate of the degree of DNA homology or genetic relatedness between two organisms can be worked out. These techniques have shown, for example, the relation between mv and animal lentiviruses. They have also been used to shed light on the evolutionary trees of various animal species. In summary, recent advances in DNA molecular biology have been exciting and suggest many potential applications in the near future. Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009). FIGURE 4 Schematic illustration to show how the DNA probe binds only to complementary DNA in the specimen DNA PROBES DNA probe specimen 1 (negative) radiolabel DNA fragment il':i:'::~ RECOMMENDED FURTHER READING specimen 2 (positive) probe binds to Scientific American 1985 Oct (Molecules of Life issue). Nussenzweig V, Nussenzweig R. Development of a sporozoite malaria vaccine. Am J Trop Med Jfyg 1986; 35: 67888. Eisenstein B. Engleberg N. Applied molecular genetics: New tools for microbiologists and clinicians. J In/ Dis 1986; 153: 41630. Somer SS, Sobell JL. Application of DNA-based diagnosis to patient care: The example of hemophilia A. Mayo Clin Proc 1987; 62: 387-404. complementary DNA strand The DNA probe,(e.g. one for EBV) is applied to a filter-immobilized clinical specimen such as throat washings or lymphocytes. If the probe remains bound, as indicated by the persistence of the label after washing, then EB virus must have been present in the specimen. The beauty of using the DNA probe approach is that it has the potential for great specificity and sensitivity. Since all living organisms contain either 211