REFLECTIONS ON ISSUES POSED BY RECOMBINANT DNA MOLECULE TECHNOLOGY. I1 Stanfield Rogers
Department of Biochemistry University of Tennessee Center for the Health Sciences Memphis, Tennessee 381 63 About 30 years ago Avery and associates discovered that DNA was the genetic material and indeed could be used t o transform one pneumococcal bacterial type t o another.’ At that time many biological scientists began t o think of using DNA in plants and animals in roughly similar ways. Fortunately or unfortunately, in terms of ethical considerations toward which this conference is basically directed, it was found that “naked” DNA administered in plants and animals was rapidly degraded by extracellular and intracellular nucleases. The best known of these nucleases are those that have been very specifically worked with by Boyer and associates. These are are the bacterial restriction enzymes, which destroy DNA molecules, which have certain sequences that the organisms recognize as being foreign.’ Some years after Avery’s discovery Lederberg and associates found, again in bacteria, that certain bacterial viruses picked up host genetic information and, upon infecting new bacteria, could transduce the genetic information from the originally infected organism t o newly infected o n e s 3 At first this was described as general transduction since the information incorporated by the virus from the host appeared rather random. Later Morse and Lederberg found that certain bacteriophages were highly selective in the host information that they i n ~ o r p o r a t e d . In ~ very recent years it has been found by Merrill in human tissue-cultured cells4 and by Doy and associates’ in tomato callus in tissue culture that lambda phage could infect and transmit genetic information for the galactose operon from E. coli to such tissue-cultured cells. About ten years ago our laboratory found that the Shope rabbit papilloma virus coded for the enzyme arginase,6 but it was really several years later before the implications of this fact became clear.7 We found that animals such as rabbits, mice and rats that were infected with this virus developed low blood arginine levels because of a systemic effect of the virus8 Then, much to our surprise, we discovered that of the people who had worked in the laboratory with this virus about half could be detected from studies of their blood arginine alone.* No other effects that were at all harmful could be found. It was clear that we had uncovered a therapeutic agent in search of a disease! Shortly after this discovery we learned of two German children who had argininemia. They had a disruption of the urea cycle because they were genetically deficient in arginase. These children were epileptic, spastic, grossly retarded, and progressively becoming worse. In view of laboratory experience that went back almost 40 years. i t seemed worthwhile to take the risk (which we had n o reason to believe existed anyway) of administering the virus to the children in the hope of replacing their genetically deficient enzyme. The mother and father of these children were heterozygotes, as were two of the other children in the family. Although their arginine levels were somewhat elevated, they had n o other
66
Rogers: Issues Posed by DNA Molecule Technology. I1
67
problems.' Since the use of virus genetic information t o replace that lost because of a deficiency disease had not been attempted before, extreme caution was used. The t w o children with argininemia were given a dose of virus that had been purified in cesium chloride and was shown by electron microscope scanning t o contain only the Shope virus. Immunological studies and blind passage in tissue-cultured cells revealed n o other virus o r harmful effects. The dose of virus first used was about 1/20th of that which we had previously found harmless t o mice. As might be expected from this tiny dose, n o effect whatever was found either in the condition of the children or in their arginine or blood ammonia levels. The high ammonia levels seemed the most likely source of their progressive disease. This virus has the advantage for these purposes of not propagating in man or even in the domestic rabbit so the amount of viral DNA involved is all the individual ever gets, About a year later the older child, whose condition had further deteriorated, was given a dose of virus about that which we would give a rabbit. A short time thereafter the blood arginine level dropped transiently, but it subsequently returned t o its original high levels. Thereafter, however, the child gradually improved. It is not certain, however, whether this was related t o the virus o r was an effect of the low protein diet, which was known to lower ammonia levels.'" However, in tissue cultures of this child's cells it had been possible t o induce arginase activity, as was shown by biochemical studies, and also t o demonstrate the presence of the enzyme in the cells by immunological means." At about this time the family had another child, who was also found t o have the disease. It seemed t o my collaborators, Terheggen in Cologne, Lowenthal in Antwerp, and Columbo in Bern, and t o myself that we might have a better chance if the child was given the virus at this younger age. Unfortunately, the difficulty t o the parents in having three sick children delayed the completion of the necessary tests for several months after the purified virus was sent to Germany. In the interim it was stored at 4°C. This virus, like many others, becomes unstable after cesium cholide purification, and when, after inoculation of the child, it was tested in rabbits and by scanning in the electron microscope, n o evidence of infectivity was found and n o intact particles were seen under the electron microscope. It was evident that the child received n o infective virus. As might be expected, there was n o effect upon the blood arginine o r ammonia levels.' Although these results were at best disappointing, it still seems to us that the chance t o prevent progressive deterioration in these children's condition was the only ethical route to take and that, should other children be found t o have this disease, they should be so treated. It is clear that one cannot hope t o be able t o find specifically sought genetic information o n various passenger viruses without making a gigantic effort. Therefore it seemed worthwhile t o try t o modify the genetic constitution of a virus in the laboratory and have this specific modification be demonstrated in its effect on infected cells. The virus of choice at the time was the tobacco mosaic virus, an RNA virus. The RNA is linear. By means of the enzyme polynucleotide phosphoralase, it is possible t o add sequentially nucleotides such as adenylic acid t o the end of the RNA molecule. This was carried out.' The RNA was found to have maintained its infectivity for tobacco plants and t o induce polylysines in the infected plants. Polyadenylic acid was known from the work of Ochoa' t o genetically code for lysine. Subsequently, the modified virus was cloned by four passages in tobacco plants and the cloned virus produced only single polylysine.I4 The cloned virus was also capable of infecting cucumbers and produced a single polylysine. Judging from the polylysine's elution characteristics o n
68
Annals New York Academy of Sciences
carboxymethyl cellulose, it appeared t o be somewhere between a septa- and a decalysine. Although the value of this modification seemed mainly theoretical, the brilliant recent work of Jackson, Symons, and Bergls demonstrated that one could add long segments of specific double-stranded DNA to a double-stranded DNA virus. In their experiment lambda phage DNA was incorporated into S.V. 40 DNA. Potentially this provides an extremely powerful tool for the treatment of disease. In spite of the ethical and scientific issues, this line of investigation should be pursued. One of the most promising possibilities of value t o mankind is the potential incorporation of nitrogen-fixing genetic information into certain plant viruses. The cauliflower mosaic virus comes immediately t o mind since it is t h e only known DNA plant virus.’ As in any such experiments foreseen and unforeseen difficulties exist. One is t o develop a virus line of very limited pathogenicity before the addition of the desired information. Another is t o find a way around the problem of the extreme oxygen-sensitivity of the nitrogenase complex of enzymes. Another problem is the requirement of the enzyme glutamine synthetase for the activation of nitrogenase synthesis.‘ The turnip may meet these conditions since its root contains large amounts of glutamine and it is hoped it is sufficiently anerobic t o eliminate the problem of oxygen-sensitivity of these enzymes. In any event it seems clear that the passage of new and needed genetic information in both plants and animals has implications that justify the calculated risk of future investigations. Guidelines are being formulated, and one suggestion is that “self-destruct” information be built into modified viruses. Interestingly, the Shope virus has a built-in mechanism of this sort. The permissiveness of the virus is dependent upon the availability of glutamine. The wild cottontail rabbit, found in certain regions of the United States, endemically carries papillomas, producing large amounts of virus. Papillomas were tested in t h e same rabbits in the laboratory t o find if their virus yield changed when the rabbits were fed a diet of commercial chow. It was found that the virus yield dropped precipitously (TABLE 1). The amino acid content of commercial chow was compared with a mixture of wheat and alfalfa, which presumably is the principle diet (e.g., in western Kansas) of the rabbit. The only striking difference
TABLE 1 COMPARISON OF VIRUS YIELD FROM PAPILLOMAS ON RABBITS O N DIFFERENT DIETS
Rabbit 1 2 3 4 5 6
7 8 9
Kansas Diet
Chow
Chow +
Diet
Glutarnine Diet
0.70 0.50 0.60 1.00 1.80 .25 -
0.40
-
-
0.25 0.10 0.30 0.25 0.08 0.15
-
-
2.0 0.25 0.25 1.90
69
Rogers: Issues Posed b y DNA Molecule Technology. I1
found was the amount of glutamine in the diets (TABLE 2). The Kansas diet had much more glutamine than did the commercial rabbit chow. It was then found (TABLE 1 ) that when wild rabbits were given glutamine parenterally, the viral yields of their papillomas greatly increased. The amount of virus was determined by extracting the papillomas, pelleting the virus in the centrifuge, and then using glycerin gradients, 5-25% t o band the virus. The amount of virus in the band was determined from its optical density. The band was then checked in the electron microscope for confirmation, as was the infectivity in rabbits. Domestic rabbits make sizable amounts of virus DNA’8 but few, if any, intact virons; b u t when a glutamine-supplemented wheat diet was used in two experiments, virus was successfully extracted from these papillomas. In these experiments quantitation was not carried out, but the lack of infective virus in domestic rabbit papillomas has been found in many laboratories over a period of many years.’ There is a mutant line of Shope virus that is weakly transmissible in domestic rabbits.” The papillomas transmitted in rabbits on the glutamine-supplemented diets were found t o be from wild-type rabbits in that they were not transmissible without glutamine supplementation. To return t o the problems of this conference per se, if it continues t o be difficult t o use viruses t o transmit genetic information, then medical progress in the treatment of many diseases will be impeded. For example, much criticism has been made of the use of the virus in argininemia despite the fact that various investigators, such as Shope and Rous and their associates, have had more than 40 years experience with the virus. In addition, the virus is not propaable without laboratory manipulation of the nutrition in any animal except the wild cottontail rabbit living o n the plains of Kansas o r in certain other areas where a high glutamine diet is available. Should the present attitudes of both social and biological scientists continue t o act t o inhibit research in this area, i t will be at
TABLE 2 COMPARISON O F AMINO ACID CONTENT (prnol%) OF COMMERCIAL CHOW AND WHEAT -
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Glutamine/100 gm
Chow
Wheat
9.86 5.48 6.72 5.74 16.64 12.14 10.37 4.45 1.41 4.42 9.13 2.65 4.24 2.25 1.44 2.98 0.59 gm
5.27 3.70 6.33 10.19 33.34 9.18 5.72 3.38 1.03 3.81) 7.16 2.42 3.86 1.23 1.14 2.18 1.26 gm
70
Annals New York Academy of Sciences
least 50 years before advantage can b e t a k e n o f the immense potential o f such studies, if we are to ju d g e f r o m the experience with the Shope virus. In t h a t period of t i m e countless individuals w h o probably could have b een helped will b e allowed to d i e o f progressive disease. F u r th e r m o r e, h u n d r ed s of t h o u san d s o f others in many parts of the world will starve because of insufficient nitrogen available to raise plants in a n improved yield. I a m particularly aw ar e of this problem because of t h e difficulty i n getting f u n d s f o r work i n this area. REFERENCES
1.
6. 7. 8.
9. 10.
AVERY, 0. T., C. M. MACLEOD, M. MC CARTY. 1944. Studies of the chemical nature
of the substance inducting transformation of pneumococcal types. 3. Exp. Med. 79: 137. GREEN, P. J . , M. C. BETLACH, H. M. GOODMAN & H. V. BOYER. The eco-restriction endonuclease. Methods Enzymol. In press. MORSE, M. L., E. M. LEDERBERG & J . LEDERBERG. 1956. Transductional heterogenotes in Escherichia coli. Genetics, 41, 758, 1956. MERRIL, C. R., M. GEIER & J. PETRICCIANL 1971. lnduction of galactose operon in human cells using lambda phage. Nature 233: 398. DOY, C. H., P. M. GRESSHOFF & B. G. ROLF. 1973. Biological and molecular evidence for transgenosis of genes from bacteria to plant cells. Proc. Nat. Acad. Sc. U.S.A. 7 0 723. ROGERS, S. 1959. Induction of arginase in rabbit epithelium by the Shope papilloma virus. Nature 183: 1815. ROGERS, S. 1971. Change in the structure of the Shope papilloma virus induced arginase associated with mutation of the virus. J . Exp. Med. 134: 1442. ROGERS, S. 1966. Shope papilloma virus: A passenger in man and its significance to the potential control of the host genome. Nature 21 2: 1220, 1966. TERHEGGEN, H. G., A. SCHWENK, M. VAN SANDE, A. LOWENTHAL & J. P. COLUMBO. 1969. Argininemia with arginase deficiency. Lancet 2: 748. COLUMBO, 3. P., H. G. TERHEGGEN, A. LOWENTHAL, M. V A N SANDE & S. ROGERS. 1473. Inborn Errors of Metabolism. Hommes and Van den Berg, Eds.
Academic Press. New York, N.Y. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
ROGERS, S., H. G. TERHEGGEN, A. LOWENT€IAL & 3 . P. COLUMBO. 1973.
Induction of arginase activity with the Shope virus in tissue cultured cells from an argininemic patient. J. Exp. Med. 137: 1091. ROGERS, S. & 1’. PFUDERER. 1968. Use of viruses as carriers of added genetic information. Nature 219: 749. KAZIHO, Y., A. GROSSMAN & S. OCHOA. 1963. Proc. Nat. Acad. Sci. U.S.A. 50: 54. ROGERS, S. 1971. Genetic engineering In Human Genetics (Proceedings of the 4th International Congress of Human Genetics, Paris). Amsterdam, The Netherlands. JACKSON, D. A., K. H. SYMONS & P. BEKG. 1972. Biochemical method for inserting new genetic information into DNA of simian virus 40. Proc. Nat. Acad. Sci. U.S.A. 69: 2904. SHEPHARD, R. J., G. E. BKUENING & R. J . WAKEMAN. 1970. Double stranded DNA from the cauliflower mosaic virus. Virology 41: 339. SHANMUGAM, K. T. & R. C. VALENTINE. 1975. Molecular biology and nitrogen fixation. Science 187: 919. ITO, Y. 1960. A tumor producing factor extracted by phenol from papillomatous tissue (Shope) of cottontail rabbits. Virology 12: 596. ROGERS, S., J . C. K l D D & P. ROUS. Relationships of the Shope virus to the cancers in domestic rabbits Acta Un. Int. Contre Cancre Vol. XVI. SELBIE, F. R., R. H. ROBERSON & R. E. SHOPE. 1953. Reversion of adaptation of Shope virus to domestic rabbits by passage through cottontails. Brit. J. Can. 2: 375.
Department of Biochemistry University of Tennessee Center for the Health Sciences Memphis, Tennessee 381 63 About 30 years ago Avery and associates discovered that DNA was the genetic material and indeed could be used t o transform one pneumococcal bacterial type t o another.’ At that time many biological scientists began t o think of using DNA in plants and animals in roughly similar ways. Fortunately or unfortunately, in terms of ethical considerations toward which this conference is basically directed, it was found that “naked” DNA administered in plants and animals was rapidly degraded by extracellular and intracellular nucleases. The best known of these nucleases are those that have been very specifically worked with by Boyer and associates. These are are the bacterial restriction enzymes, which destroy DNA molecules, which have certain sequences that the organisms recognize as being foreign.’ Some years after Avery’s discovery Lederberg and associates found, again in bacteria, that certain bacterial viruses picked up host genetic information and, upon infecting new bacteria, could transduce the genetic information from the originally infected organism t o newly infected o n e s 3 At first this was described as general transduction since the information incorporated by the virus from the host appeared rather random. Later Morse and Lederberg found that certain bacteriophages were highly selective in the host information that they i n ~ o r p o r a t e d . In ~ very recent years it has been found by Merrill in human tissue-cultured cells4 and by Doy and associates’ in tomato callus in tissue culture that lambda phage could infect and transmit genetic information for the galactose operon from E. coli to such tissue-cultured cells. About ten years ago our laboratory found that the Shope rabbit papilloma virus coded for the enzyme arginase,6 but it was really several years later before the implications of this fact became clear.7 We found that animals such as rabbits, mice and rats that were infected with this virus developed low blood arginine levels because of a systemic effect of the virus8 Then, much to our surprise, we discovered that of the people who had worked in the laboratory with this virus about half could be detected from studies of their blood arginine alone.* No other effects that were at all harmful could be found. It was clear that we had uncovered a therapeutic agent in search of a disease! Shortly after this discovery we learned of two German children who had argininemia. They had a disruption of the urea cycle because they were genetically deficient in arginase. These children were epileptic, spastic, grossly retarded, and progressively becoming worse. In view of laboratory experience that went back almost 40 years. i t seemed worthwhile to take the risk (which we had n o reason to believe existed anyway) of administering the virus to the children in the hope of replacing their genetically deficient enzyme. The mother and father of these children were heterozygotes, as were two of the other children in the family. Although their arginine levels were somewhat elevated, they had n o other
66
Rogers: Issues Posed by DNA Molecule Technology. I1
67
problems.' Since the use of virus genetic information t o replace that lost because of a deficiency disease had not been attempted before, extreme caution was used. The t w o children with argininemia were given a dose of virus that had been purified in cesium chloride and was shown by electron microscope scanning t o contain only the Shope virus. Immunological studies and blind passage in tissue-cultured cells revealed n o other virus o r harmful effects. The dose of virus first used was about 1/20th of that which we had previously found harmless t o mice. As might be expected from this tiny dose, n o effect whatever was found either in the condition of the children or in their arginine or blood ammonia levels. The high ammonia levels seemed the most likely source of their progressive disease. This virus has the advantage for these purposes of not propagating in man or even in the domestic rabbit so the amount of viral DNA involved is all the individual ever gets, About a year later the older child, whose condition had further deteriorated, was given a dose of virus about that which we would give a rabbit. A short time thereafter the blood arginine level dropped transiently, but it subsequently returned t o its original high levels. Thereafter, however, the child gradually improved. It is not certain, however, whether this was related t o the virus o r was an effect of the low protein diet, which was known to lower ammonia levels.'" However, in tissue cultures of this child's cells it had been possible t o induce arginase activity, as was shown by biochemical studies, and also t o demonstrate the presence of the enzyme in the cells by immunological means." At about this time the family had another child, who was also found t o have the disease. It seemed t o my collaborators, Terheggen in Cologne, Lowenthal in Antwerp, and Columbo in Bern, and t o myself that we might have a better chance if the child was given the virus at this younger age. Unfortunately, the difficulty t o the parents in having three sick children delayed the completion of the necessary tests for several months after the purified virus was sent to Germany. In the interim it was stored at 4°C. This virus, like many others, becomes unstable after cesium cholide purification, and when, after inoculation of the child, it was tested in rabbits and by scanning in the electron microscope, n o evidence of infectivity was found and n o intact particles were seen under the electron microscope. It was evident that the child received n o infective virus. As might be expected, there was n o effect upon the blood arginine o r ammonia levels.' Although these results were at best disappointing, it still seems to us that the chance t o prevent progressive deterioration in these children's condition was the only ethical route to take and that, should other children be found t o have this disease, they should be so treated. It is clear that one cannot hope t o be able t o find specifically sought genetic information o n various passenger viruses without making a gigantic effort. Therefore it seemed worthwhile t o try t o modify the genetic constitution of a virus in the laboratory and have this specific modification be demonstrated in its effect on infected cells. The virus of choice at the time was the tobacco mosaic virus, an RNA virus. The RNA is linear. By means of the enzyme polynucleotide phosphoralase, it is possible t o add sequentially nucleotides such as adenylic acid t o the end of the RNA molecule. This was carried out.' The RNA was found to have maintained its infectivity for tobacco plants and t o induce polylysines in the infected plants. Polyadenylic acid was known from the work of Ochoa' t o genetically code for lysine. Subsequently, the modified virus was cloned by four passages in tobacco plants and the cloned virus produced only single polylysine.I4 The cloned virus was also capable of infecting cucumbers and produced a single polylysine. Judging from the polylysine's elution characteristics o n
68
Annals New York Academy of Sciences
carboxymethyl cellulose, it appeared t o be somewhere between a septa- and a decalysine. Although the value of this modification seemed mainly theoretical, the brilliant recent work of Jackson, Symons, and Bergls demonstrated that one could add long segments of specific double-stranded DNA to a double-stranded DNA virus. In their experiment lambda phage DNA was incorporated into S.V. 40 DNA. Potentially this provides an extremely powerful tool for the treatment of disease. In spite of the ethical and scientific issues, this line of investigation should be pursued. One of the most promising possibilities of value t o mankind is the potential incorporation of nitrogen-fixing genetic information into certain plant viruses. The cauliflower mosaic virus comes immediately t o mind since it is t h e only known DNA plant virus.’ As in any such experiments foreseen and unforeseen difficulties exist. One is t o develop a virus line of very limited pathogenicity before the addition of the desired information. Another is t o find a way around the problem of the extreme oxygen-sensitivity of the nitrogenase complex of enzymes. Another problem is the requirement of the enzyme glutamine synthetase for the activation of nitrogenase synthesis.‘ The turnip may meet these conditions since its root contains large amounts of glutamine and it is hoped it is sufficiently anerobic t o eliminate the problem of oxygen-sensitivity of these enzymes. In any event it seems clear that the passage of new and needed genetic information in both plants and animals has implications that justify the calculated risk of future investigations. Guidelines are being formulated, and one suggestion is that “self-destruct” information be built into modified viruses. Interestingly, the Shope virus has a built-in mechanism of this sort. The permissiveness of the virus is dependent upon the availability of glutamine. The wild cottontail rabbit, found in certain regions of the United States, endemically carries papillomas, producing large amounts of virus. Papillomas were tested in t h e same rabbits in the laboratory t o find if their virus yield changed when the rabbits were fed a diet of commercial chow. It was found that the virus yield dropped precipitously (TABLE 1). The amino acid content of commercial chow was compared with a mixture of wheat and alfalfa, which presumably is the principle diet (e.g., in western Kansas) of the rabbit. The only striking difference
TABLE 1 COMPARISON OF VIRUS YIELD FROM PAPILLOMAS ON RABBITS O N DIFFERENT DIETS
Rabbit 1 2 3 4 5 6
7 8 9
Kansas Diet
Chow
Chow +
Diet
Glutarnine Diet
0.70 0.50 0.60 1.00 1.80 .25 -
0.40
-
-
0.25 0.10 0.30 0.25 0.08 0.15
-
-
2.0 0.25 0.25 1.90
69
Rogers: Issues Posed b y DNA Molecule Technology. I1
found was the amount of glutamine in the diets (TABLE 2). The Kansas diet had much more glutamine than did the commercial rabbit chow. It was then found (TABLE 1 ) that when wild rabbits were given glutamine parenterally, the viral yields of their papillomas greatly increased. The amount of virus was determined by extracting the papillomas, pelleting the virus in the centrifuge, and then using glycerin gradients, 5-25% t o band the virus. The amount of virus in the band was determined from its optical density. The band was then checked in the electron microscope for confirmation, as was the infectivity in rabbits. Domestic rabbits make sizable amounts of virus DNA’8 but few, if any, intact virons; b u t when a glutamine-supplemented wheat diet was used in two experiments, virus was successfully extracted from these papillomas. In these experiments quantitation was not carried out, but the lack of infective virus in domestic rabbit papillomas has been found in many laboratories over a period of many years.’ There is a mutant line of Shope virus that is weakly transmissible in domestic rabbits.” The papillomas transmitted in rabbits on the glutamine-supplemented diets were found t o be from wild-type rabbits in that they were not transmissible without glutamine supplementation. To return t o the problems of this conference per se, if it continues t o be difficult t o use viruses t o transmit genetic information, then medical progress in the treatment of many diseases will be impeded. For example, much criticism has been made of the use of the virus in argininemia despite the fact that various investigators, such as Shope and Rous and their associates, have had more than 40 years experience with the virus. In addition, the virus is not propaable without laboratory manipulation of the nutrition in any animal except the wild cottontail rabbit living o n the plains of Kansas o r in certain other areas where a high glutamine diet is available. Should the present attitudes of both social and biological scientists continue t o act t o inhibit research in this area, i t will be at
TABLE 2 COMPARISON O F AMINO ACID CONTENT (prnol%) OF COMMERCIAL CHOW AND WHEAT -
Aspartic acid Threonine Serine Proline Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Glutamine/100 gm
Chow
Wheat
9.86 5.48 6.72 5.74 16.64 12.14 10.37 4.45 1.41 4.42 9.13 2.65 4.24 2.25 1.44 2.98 0.59 gm
5.27 3.70 6.33 10.19 33.34 9.18 5.72 3.38 1.03 3.81) 7.16 2.42 3.86 1.23 1.14 2.18 1.26 gm
70
Annals New York Academy of Sciences
least 50 years before advantage can b e t a k e n o f the immense potential o f such studies, if we are to ju d g e f r o m the experience with the Shope virus. In t h a t period of t i m e countless individuals w h o probably could have b een helped will b e allowed to d i e o f progressive disease. F u r th e r m o r e, h u n d r ed s of t h o u san d s o f others in many parts of the world will starve because of insufficient nitrogen available to raise plants in a n improved yield. I a m particularly aw ar e of this problem because of t h e difficulty i n getting f u n d s f o r work i n this area. REFERENCES
1.
6. 7. 8.
9. 10.
AVERY, 0. T., C. M. MACLEOD, M. MC CARTY. 1944. Studies of the chemical nature
of the substance inducting transformation of pneumococcal types. 3. Exp. Med. 79: 137. GREEN, P. J . , M. C. BETLACH, H. M. GOODMAN & H. V. BOYER. The eco-restriction endonuclease. Methods Enzymol. In press. MORSE, M. L., E. M. LEDERBERG & J . LEDERBERG. 1956. Transductional heterogenotes in Escherichia coli. Genetics, 41, 758, 1956. MERRIL, C. R., M. GEIER & J. PETRICCIANL 1971. lnduction of galactose operon in human cells using lambda phage. Nature 233: 398. DOY, C. H., P. M. GRESSHOFF & B. G. ROLF. 1973. Biological and molecular evidence for transgenosis of genes from bacteria to plant cells. Proc. Nat. Acad. Sc. U.S.A. 7 0 723. ROGERS, S. 1959. Induction of arginase in rabbit epithelium by the Shope papilloma virus. Nature 183: 1815. ROGERS, S. 1971. Change in the structure of the Shope papilloma virus induced arginase associated with mutation of the virus. J . Exp. Med. 134: 1442. ROGERS, S. 1966. Shope papilloma virus: A passenger in man and its significance to the potential control of the host genome. Nature 21 2: 1220, 1966. TERHEGGEN, H. G., A. SCHWENK, M. VAN SANDE, A. LOWENTHAL & J. P. COLUMBO. 1969. Argininemia with arginase deficiency. Lancet 2: 748. COLUMBO, 3. P., H. G. TERHEGGEN, A. LOWENTHAL, M. V A N SANDE & S. ROGERS. 1473. Inborn Errors of Metabolism. Hommes and Van den Berg, Eds.
Academic Press. New York, N.Y. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
ROGERS, S., H. G. TERHEGGEN, A. LOWENT€IAL & 3 . P. COLUMBO. 1973.
Induction of arginase activity with the Shope virus in tissue cultured cells from an argininemic patient. J. Exp. Med. 137: 1091. ROGERS, S. & 1’. PFUDERER. 1968. Use of viruses as carriers of added genetic information. Nature 219: 749. KAZIHO, Y., A. GROSSMAN & S. OCHOA. 1963. Proc. Nat. Acad. Sci. U.S.A. 50: 54. ROGERS, S. 1971. Genetic engineering In Human Genetics (Proceedings of the 4th International Congress of Human Genetics, Paris). Amsterdam, The Netherlands. JACKSON, D. A., K. H. SYMONS & P. BEKG. 1972. Biochemical method for inserting new genetic information into DNA of simian virus 40. Proc. Nat. Acad. Sci. U.S.A. 69: 2904. SHEPHARD, R. J., G. E. BKUENING & R. J . WAKEMAN. 1970. Double stranded DNA from the cauliflower mosaic virus. Virology 41: 339. SHANMUGAM, K. T. & R. C. VALENTINE. 1975. Molecular biology and nitrogen fixation. Science 187: 919. ITO, Y. 1960. A tumor producing factor extracted by phenol from papillomatous tissue (Shope) of cottontail rabbits. Virology 12: 596. ROGERS, S., J . C. K l D D & P. ROUS. Relationships of the Shope virus to the cancers in domestic rabbits Acta Un. Int. Contre Cancre Vol. XVI. SELBIE, F. R., R. H. ROBERSON & R. E. SHOPE. 1953. Reversion of adaptation of Shope virus to domestic rabbits by passage through cottontails. Brit. J. Can. 2: 375.
