Gene therapy in Drosophila

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T I B S - J u n e 1 983 continuedfrom p 190

understood. In some cases the gene is qualitatively changed, for example the transcripts of the c - m y c oncogene in mouse plasmacytomas and Burkitt's lymphoma tend to be shorter than the normal product 8 and oncogene activation in human bladder carcinoma is associated with a single point mutation8.lo. However, increased transcription of the normal cellular homologue of transforming oncogenes can also induce tumours. For example, transformation of recipient ceils has been shown by the normal counterparts of mouse c - m o s oncogene and rat and human c - H a - r a s l oncogenes, provided that a viral long terminal repeat sequence is ligated upstream of the gene n,~. Accumulating evidence of this kind supports the view that some cancers may arise from a change in the transcriptional control of oncogenes induced by viral or other mechanisms, such as somatic rearrangement. Translocations and deletions, leading to new locations of oncogenes, are clearly associated with malignant transformation of many tissues ~and this too is consistent with oncogenes being released from transcriptional control or coming under the control of a new promoter. There are various ways in which incorporation of mtDNA sequences into the nuclear genome might activate oncogenes: (1)

insertion into a regular nucleosomic region at a critical point might alter the genomic conformation, encouraging breakpoints and somatic rearrangement; (2) insertion into the gene itself would be mutagenic; (3) insertion of mtDNA containing an origin of transcription might effectively bring an oncogene under the control of a new promotor. Blanc and Dujon TM have recently shown that replication sequences active in vivo in yeast mitochondria can drive the autonomous replication of recombinant plasmids, demonstrating that they can act as replication origins in the cell outside the mitochondria, a feature of some interest in relation to oncogene amplification ~4. In fact, the various possible mechanisms discussed by Galloway and McDougall ~5 for transformations induced by Herpes simplex DNA may be equally valid for migratory mtDNA, including 'hit and run' mechanisms where the retention of inserted DNA is not necessary to maintain transformation. Despite the lack of direct evidence for a mitochondrial role in carcinogenesis current trends in molecular genetics indicate that this area should not be overlooked.

References 1 Rowley, J. D. (1983)Nature 301,290-291 2 Neel, B. G., Jhanwar, S. C., Chaganti, R. S. K. and Hayward, W. S. (1982 ) Proc. Natl Acad. Sci. USA 79, 7842-7846

Gene therapy in Drosophila An example of successful 'gene therapy' has been recently described by G. Rubin and A. Spradling t. They report that the introduction of DNA carrying a wild-type copy of the rosy gene into Drosophila carrying a mutation at the rosy locus restored the normal phenotype. Their success is based upon the development of a transformation system which allows introduction of new genetic information into the Drosophila germline. In their study of Drosophila transposable elements, Rubin and his collaborators became interested in a syndrome of genetic traits called hybrid dysgenesis. When certain Drosophila strains are interbred, high rates of sterility, mutation and chromosomal aberration are seen. These traits occur only when males of'P' or paternally contributing strains are bred to females of 'M' or maternally contributing strains, but not when the reciprocal cross is performed. The high reversion rate of the resulting mutations suggested they might be caused by DNA insertions. The stability of these mutations also depended upon the genetic environment - reversion was not seen when the mutation was maintained in P strains, but occurred frequently in M

strains. P strains have been shown to contain, in their genome, a family of transposable DNA elements, called P-factors. Using a cloned 'white' gene (the wild-type version of a gene affecting eye color) Rubin, Kidwell and Bingham2 analysed the structure of the DNA at the white locus in several white mutants which arose in dysgenic crosses. Of seven independent mutants, five were shown to result from the insertion of a P-element into the white gene. The remaining two mutants were caused by insertion of a different Drosophila transposable element, copia. The mutations resulting from P-element insertions were stable in P genetic environments, but reverted at high frequency when placed in an M background. Reversion was caused by excision of the P-element, restoring the normal activity of the gene. In contrast, the mutations resulting from copia insertions were stable in both genetic environments. Thus, they concluded that hybrid dysgenesis occurs because P elements present in P strain pronuclei transpose at high frequency when in M strain eggs, but seldom, if at all when in P strain eggs. Apparently the P strains contain something which represses P ele-

3 Farrelly,F. and Butow, R. A. (1983)Nature 301, 296-301 4 Gellissen,G., Bradfield,J. Y., White, B. N. and Wyatt, G. R. (1983)Nature 301,631-634 5 Jacobs, H. T., Posakony, J. W. and Davidson, E. H. J. Mol. Biol. (in press) 6 Wright, R. M. and Cummings, D. J. (1983) Nature 302, 86-88 7 Wilkie,D. and Evans,1. (1982)Trends Biochem. Set. 7, 147-151 8 Marcu,K. B., Hams, L. J., Stanton,L. W., Erikson, J., Watt, R. and Croce, C. M. (1983)Proc. Natl Acad. Sci. USA 80, 519-523 9 Reddy, E. P., Reynolds, R. K., Santos, E. and Barbacid,M. (1982)Nature 300, 149-152 10 Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg,P. H. and Goeddel, D. V. (1983)Nature 302, 33-37 11 Blair, D. G., Oskarsson, M., Wood, T. G., McClements, W. L., Fischinger,P. J. and Vande Wonde, G. G. (1981)Science 212, 941-943 12 Chang,E. H., Furth, M. E., Scolnick, E. M. and Lowy, D. R. (1982)Nature 297,479--483 13 Blanc,H. and Dujon, B. (1982)in Mitochondrial Genes, pp. 279--294, Cold Spring Harbor Laboratory 14 McCoy, M. S., Toole, J. J., Cunningham,J. M., Chang, E. H., Lowy, D. R. and Weinberg,R. A. (1983)Nature 302, 79-81 15 Galloway, D. A. and McDougal], J. K. (1983) Nature 302, 21-24 ROBERT A. REID Department of Biology, University of York, York YOI 5DD, UK.

ment transposition, while M strains do not. Two types of P-element are observed in P-strain flies, One is about 3 kb in length, has 31 bp inverted terminal repeats and probably represents a 'complete' copy of the element. Complete P-elements have three open reading frames suggesting they could encode the enzymes necessary for transposition. The other type are smaller, but retain the terminal repeats, suggesting that sequences internal to the element are missing. Based on the genetic data, Spradling and Rubin s wondered if a cloned P-element would transpose when introduced into an M-strain embryo, To identify such a transposition event, they took advantage of a genetic marker, s i n g e d - w e a k , which had arisen during a dysgenic cross from the insertion of a deleted type P-element into the singed locus. When females of the mutant strain are mated to P strain males, only 50% of the progeny display the singed-w phenotype. The remainder are split between an easily observed more severe version of the phenotype and wild type. Rubin and Spradling believe this occurs because the deleted P-element is incapable of catalysing its own transposition, presumably because it does not encode its own transposase. It can, however, respond to transposase produced

~t'~1983, ElsevierScience Publishers BV. Amsterdam 0376 - 5067/83/$01 00

192 in P-strains, probably by the complete P-elements. Thus, to observe transposition following introduction of a complete P-element into s i n g e d - w mutants, they simply looked for a change in the singed phenotype. In flies with altered phenotype, they argue that the deleted P-elements have been induced to transpose just as they would in a dysgenic cross. Microinjection of the cloned P-element into embryos resulted in as many as 48% of the fertile offspring displaying mutability at the s i n g e d locus. In addition, the second generation males, when mated to the appropriate females, showed continued mutability suggesting they now carried a complete P-element capable of providing transposase. Southern blot analysis confirmed that the P-element was integrated by a bona-fide transposition event, because only P-element DNA was seen. I n - s i t u hybridization showed that the 3 kb P-element had indeed become integrated into the chromosome. Having demonstrated that a cloned P-element would transpose itself into the genome of a microinjected embryo, Rubin and Spradling ~ asked if a gene inserted into the cloned P-element would be carried along when transposition occurred. They introduced the gene for xanthine dehydrogenase - the r o s y locus - into a cloned, deleted type P element to generate a r o s y transposon. Then following co-injection of the rosy transposon and a 3 kb P-element into an embryo carrying a rosy mutation they screened the progeny of the injected embryo for wild-type eye color. As many as half the progeny were found to show the wild-type eye color and in subsequent generations the wild-type eye color was inherited stably. As for the injected 3 kb P-elements mentioned earlier, only sequences between the 31 bp terminal repeats were inserted into the genome, again confm-ning that integration was occurring via transposition. The widespread occurrence of repetitive DNA having transposon-like structures similar to those seen in P-elements suggests that similar mechanisms might work in

TIBS -June

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Fig. 1. Hybrid dysgenesis is a syndrome o f mutations which occurs because P elements present in paternal DNA are activated to transpose into new locations following its introduction into the M-strain cellular environment. Insertion o f P elements at new sites often results in insertional inactivation o f genes. Fig. 2. A cloned copy o f the wild-type rosy gene is inserted into a cloned P element. This'rosy transposon' is microinjected into a Drosophila embryo carrying the rosymutation. Transposase synthesized by the injected P element catalyses transposition o f the rosy gene from the injected DNA into the host chromosome where it can function to correct the mutation carried by' the embryo.

1983

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193

TIBS -June 1983 other organisms. As Rubin and Spradling point out, the Drosophila P-element could function in any organism where the appropilate transposase function could be provided. Genetic modification of the germline of Drosophila, with high efficiency, adds yet another powerful tool to

the arsenal available to biologists to study gene regulation during development. We can be sure that we'll soon be hearing about similar experiments in other organisms.

REX L. CHISHOLM

References 1 Rubin, G. M. and Spradling, A. C. (1982)Science 218,348-353

The earliest biochemistry C. U. M. Smith Did antiquity have a biochemistry? Certainly not in the modern sense of that term; biochemistry, as we know it, hardly existed even a century ago t. The ancient paradigm was very different from ours: the idea of inert matter being foreign to most of the presocratics 2. Nevertheless, through the distorting lens of a radically different set of presuppositions, the outlines of the modern understanding can be dimly discerned. Montalenti once entitled a short account of the history of biology: From Aristotle to Democritus via Darwin3; this might easily be adapted in the context of biochemistry to read 'From Heraclitus to Heraclitus via Dalton'. That we never step into the same river twice 4, that 'all things are in motion all the time, but that this escapes our perception 's , that equilibrium emerges from a balance of opposing forces'; this appreciation of the nature of things is as clear to the biochemically informed as it ever was to the gloomy philosopher of Ephesus (c. 500 BC). But if antiquity can hardly be said to have had a biochemistry there is no doubt that it had a biotechnology. From the remotest beginnings of Middle Eastern civilization the making of bread, beer and wine; the extraction and concentration of dyes and perfumes; the preservation of food and drink; the processes of mummification and embalming; the treatment of disease: all had flourished and all involved techniques which we now recognize as based on biochemistry and/or to involve practices basic to biochemistry. The classical Greeks recognized the immense history of their biotechnology. Furthermore, unlike their predecessors in the great river-valley civilizations of the Middle East, they imagined a natural rather than a supernatural origin and development. The writer of the Hippocratic treatise De prisca medicina gives the following account: '1 hold that not even the mode of living and nourishment enjoyed at present by men in health (\ U. M. Smith is at the Department of Biological Sciences, University o f Aston in Birmingham, Birmingham B4 7ET, UK.

2 Rubin, G. M., Kidwell, M. G. and Bingham, P. M. (1982) Cell 29,987-994 3 Spradling A. C. and Rubin, G. M. (1982) Science 218,341-347

would have been discovered, had a man been satisfied with the same food and drink as satisfy an ox, a horse, and every animal save man, for example the products of the earth - fruits, wood and grass . . . . Yet I am of the opinion that in the beginning man also used this sort of nourishment. Our present ways of living have, I think, been discovered and elaborated during a long period of time. For many and terrible were the sufferings of men from strong and brutish living when they partook of crude foods, uncompounded and possessing great powers . . . . For this reason the ancients, too, it seems to me have sought for nourishment that harmonised with their constitution and to have discovered that which we use now. So from wheat, after steeping it, winnowing, grinding and sifting, kneading, baking, they produced bread, and from barley they produced cake. Experimenting with food they boiled or baked, after mixing, many other things. . . . The discovery was a great one implying much investigation and art." Biotechnology is truly a stone-age invention ! The making of alcoholic drink may well be the most ancient of all biotechnologies. Indeed one of the few words which have survived from the earliest Sumerian script is the original form of the word 'alcohol 's . Even earlier than Sumer, however, in the Upper Palaeolithic age, well-known rock paintings show honey being collected from the hives of wild bees s. The honey, early man's sole concentrated source of sugar, was allowed to ferment to form a moderately alcoholic mead. The reputation of this early mead resonates through the millennia as 'the nectar of the gods'. With the onset of the Neolithic revolution in the Middle East, some ten to twelve thousand years ago, other fermentations became possible. By the time that the first great civilizations of antiquity originated (Sumer, c. 4000 BC; Egypt, c. 3200 BC) various forms of wine were well known. A precursor species of the modern vine ( Vitis vinifera) once grew wild over much of southern Europe and the Middle East. Grape-wine is thus very ancient. It is recorded in the archives of proto-dynastic Egypt, more than five thousand years ago TM. Egyptian paintings, carvings and texts, which the hot desert climate has preserved, give many details of its production. c

Department of Biology, MIT, Cambridge, MA 02139, USA.

The fermentation was, at first, due to yeasts which grew on the skins of the grapes. After harvesting, the grapes were crushed by treading - and later by the invention of bag presses. After fermentation the earliest pictures show the wine being drunk through lengthy tubes to avoid disturbing the sediment. Later, wine-makers are shown straining the wine through linen into jars, which were carefully sealed to prevent further oxidation by the acetic-acid microorganism, Mycodermata aceti. The sealed jars were stored in cool places and their contents usually mixed or diluted with water before being drunk. If the wine jars were not sealed further oxidation quickly occurred, especially in the 40°C temperatures of Egypt. The vinegar so formed was the strongest acid known to the ancient world and was often used for medicinal purposes or as a solvent for drugs and herbs. Grape-wine was more expensive, and consequently less popular, than either date-wine or beer. The production of date-wine is shown in Egyptian wallpaintings dating back to the Middle Kingdom, about 1900 BC. The paintings give a comprehensive and easily understood account of the production process. We see the pounding, mashing and crushing of the dates; sieving, straining and pressing of the mash; clarification by sedimentation; decanting; storage in stoppered vessels to prevent a second fermentation to acetate. All these processes had been invented by the second millennium BC. Beer seems to have been the most common drink in both ancient Sumer and Egypt. The technique of brewing once again dates back to the beginning of recorded history and the earliest texts refer to beers produced from barley and other cereal plants. It was just as welcome then as it is today, being referred to as 'the plentiful, the joy-bringer, the addition to the meal, the heavenly, the beautiful-good' (Ref. 10, p. 278n). Malting, the essential process, seems to have been transferred to the brewery from a yet more ancient biotechnology. The cereal plants which formed the food base of the earliest civilizations could be made much more palatable if the grain was forced to germinate by soaking in salty water. This also enhanced its nutritional value because, during germination, diastase converts starch to maltose,

1983.ElsevierSciencePublishersBV . Amsterdam 0376 5067/83/$0100