National Academies Press: OpenBook

Biotechnology in China (1989)

Chapter: Appendix E: Statement by Dr. Niu

« Previous: Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA
Suggested Citation:"Appendix E: Statement by Dr. Niu." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 97
Suggested Citation:"Appendix E: Statement by Dr. Niu." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 98
Suggested Citation:"Appendix E: Statement by Dr. Niu." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 99
Suggested Citation:"Appendix E: Statement by Dr. Niu." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
×
Page 100
Suggested Citation:"Appendix E: Statement by Dr. Niu." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 101
Suggested Citation:"Appendix E: Statement by Dr. Niu." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 102

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EStatement by Dr. Niu Observation on the Evaluation of Dr. Niu's Research at Ide Published in "Biotechnology in China" by Drs. Hamer and Kung There are several points in Drs. Hamer and Kung's evaluation pages upon which I would like to comment: 1. Based on the national competition for research grant, IDB got 1.8 million yuan from NSFC for 3 years and not for one year. 2. About the working hypothesis (my theory): The stored mRNA in the presence of reverse transcriptase (RT) is transcribed into cDNA which is then inserted into the genome. This cDNA would act as an agent for the differential gene activation during embryogensis. Drs. Hamer and Kung wrote "This theory directly contradicts the basic laws of Mendelian inheritance." As a developmental biologist, I appreciate fully what T.H. Morgen wrote in his book entitled Embryology and Genetics that "during cleavage heterogeneous cytoplasm modifies nuclear function." The question often being asked is what is the component (agent) of the heterogeneous cytoplasm? In the seventies we proposed that the egg mRNA transcript (cDNA) was the agent. Now in the later half of the eighties we have provided evidence to support that proposal. We do not see how it contradicts Mendel's laws. As to the transmission of the induced simple tail of goldfish to the next generation, we have reported on the phenomenon. 3. Hamer and Kung stated that the rabbit globin mRNA used in our experiment was impure. This was wrong and it strongly indicates that they did not read the STATEMENT BY DR. NIU 97

paper. In the abstract of the article we thanked Prof. Jerry Lingrel for the gift of rabbit globin mRNA (it was the best available at the time, 1978-1988). In order to show that red blood cells (RBC) from the rabbit globin mRNA injected fish carry the properties of rabbit RBC, we chose to detect rabbit globin from the fish's RBC by immuno-precipitation (highly specific) and by the tissue specific lactate dehydrogenase (LDH). The electrophoretic pattern of the liver homogenates from the injected fish was of the hybrid type (i.e., intermediate between goldfish and rabbit). Therefore, for our purpose, there was no need to do globin chemistry as demanded by Hamer and Kung. 4. Their comments about the "green fish" were totally invented. No such comments were made. Drs. Hamer and Kung labelled me as a poor professor without funding, unable to attract students or post-doctors. In view of my retirement, Drs. Hamer and Kung should know university rules governing retired professors. As a matter of fact, Temple University treats me very well, as evidenced by not closing my lab. As to my publications, Drs. Hamer and Kung should get a list of my publications in Chinese and Western journals, get to know the content and evaluate those papers published while working in IDB (1982-1990) and not only those from Institute of Zoology (1973-1981). Drs. Hamer and Kung did not know that the late Prof. Tung suggested to me that he would like to see my research done in China and published in China. In the past few years, however, Academia Sinica encourages people to publish abroad. This is the reason why I began to get papers published in Western Journals in the past few years. The evaluation was supposed to be on papers published by members of IDB. Drs. Hamer and Kung chose to comment only papers published in the period of 1973-1981. Their error was in not evaluating the work done at the IDB during the period 1982-1990; however, Drs. Hamer and Kung did write a sentence about Dr. Niu's work on cytoplasmic DNA synthesis and the discovery of an independent reverse transcriptase in the mid-1980's. Dr. Niu does express great appreciation to Dr. Hans Ris, a well-known biologist, for his letter of support and criticism therein of the Drs. Hamer and Kung's false statements. Drs. Hamer and Kung wrongfully drew the conclusion that Dr. Niu's group controls the institute's resources, positions, and power. Therefore, the research conducted by this group is open to question. Dr. Niu's scientific and professional response to these irresponsible statements was an offer already made for the impartial selection of a highly respected, qualified member of the world scientific community to come to the Institute at Beijing. All expenses would be paid and the scientist would have the permission of the IDB to monitor and see the results of the research conducted by Dr. Niu and to write an independent report of his findings. The offer by Dr. Niu was refused. Obviously, the writers are apprehensive of finding a failure to corroborate their report. STATEMENT BY DR. NIU 98

Response to Prof. Davidson's Comments on M.C. Niu's Scientific Research in Academia Sinica, Beijing, China Prof. Davidson subscribes to the dogma that transformation in developing systems is based on DNA and never on RNA. He also accepts the oncogenic effect of certain viral RNA based on the presence of the reverse transcriptase (RT for short) in the virus. In eggs, he argues, RT has not been demonstrated. Prof. Davidson, in 1989-1990, was not familiar with the work of Niu at IDB after 1982 and presents an excerpt from his manuscript written in 1981. There are seven points in Prof. Davidson's analysis of Niu's work in collaboration with the late Prof. T.C. Tung at the Institute of Zoology in Beijing. I shall answer them point by point. 1. The method of our isolation of mRNA is unreliable because of the use of "Sigmacell" type 38 cellulose. Answer: New methodology in research develops year after year. By 1972 there were 3 new techniques in the literature for the isolation of mRNA: (1) Absorption to millipore (see Lee et al., PNAS, 68:1331, 1971 and Rosenfeld et al., B. B. Res. Commun., 47:787, 1972), (2) mRNA hybridization to the oligo dT cellulose (Aviv et al., PNAS, 69:1406, 1972), and (3) isolation of eukaryotic mRNA on cellulose and its translation in vitro (Schultz et al., B. B. Res. Commun., 16:377, 1972). The method of using "Sigmacell" cellulose for mRNA isolation is simple and easy. The mRNA isolated with this technique was shown to be pure and could prime the translation of a specific protein. The reason we chose "Sigmacell" cellulose was mainly economical. It goes without saying that in Philadelphia we used ''sigmacell" cellulose first and then purified the mRNA further by oligo dT cellulose. The mRNA activity was tested by in vitro synthesis of a specific protein. 2. The mRNA and DNA mediated tail transformation from the double 2- lobed tail to the forked shaped simple goldfish tail is questionable. (Correction: 100 o.d./ml DNA is misprint of 10 o.d./ml) Answer: The claim of nucleic acid-mediated genetic change was sensational in early 1970. Many biologists were skeptical because they claimed that the strain of the goldfish we used was not genetically pure. Another criticism was that we did not use any controls. However we had used fish liver mRNA, rRNA, and tumor mRNA as controls and eggs injected with these mRNAs developed into fish without changed tails. They did not accept that mRNA could produce genetic change. This is to say that the scientific community demands that we repeat the experiments. We decided to use mRNA from a different species. When egg mRNA from a carp, Cyprinus carpio L. was injected into goldfish eggs of a pure strain maintained by inbreeding at the Institute of Zoology in Beijing, the tail was changed but with slightly reduced frequency. (Literature says that cloned rabbit globin gene (Ca. 600 b.p.) and many other genes with less than 1,000 b.p. can mediate genetic change.) STATEMENT BY DR. NIU 99

3. The transformation of the mRNA and DNA induced goldfish tail to next generation. Answer: Published observation (Matui, J. Imp. Fisheries Institute, 30:1, 1934) and our own data show that the tail of the offspring from the cross of goldfish and crucian carp or carp is simple and never double 2- lobed. The double 2-lobed tail is recessive. The injection of carp mRNA into goldfish eggs resulted in the alteration of the tail fin, while injection of goldfish egg mRNA into carp eggs provided no change of tail fin. These data show the specificity of the carp egg mRNA in goldfish tail change. The mating of the simple tailed goldfish obtained from the injected eggs produced offspring with simple forked and double 2-lobed tails, thus showing the transmission of the induced simple tail to the next generation. This refutes Prof. Davidson's assumption that the simple tail in goldfish is a minor developmental abnormality. The strain of goldfish we used is genetically pure. Their mating produced double fancy tailed offspring. 4. The newt DNA induced development of the balancer in goldfish larvae. Answer: Balancers are transitional organs found in the California newt, Taricha torosa. In newt larvae, they emerge 5-6 days after fertilization and resorb ca. 25 days later. They consist of a pair of bar-like organs on the site post-lateral to the mouth. In sections they consist of epidermis and connective tissue. The cells at the top are secretory in nature. When Taricha torosa DNA was injected into goldfish eggs, approximately 1 percent of the larvae developed one balancer on the right or left side. If DNA from the Chinese newt, Cynops orientalis, was injected into goldfish eggs, 6 percent of the larvae developed balancer. Over 90 percent of the injected eggs developed normally. Shi et al. (Scientia Sinica, 24:402-406, 1980) repeated this experiment using DNA isolated from frog and Chinese newt. Over 90 percent of their injected eggs developed abnormally. Among the abnormal tadpoles, 0.9 × 10-3 of the frog DNA and 0.3 × 10-2 percent of the newt DNA-injected developed lateral protuberance on the operculum. Without considering the definition of the newt's balancer, they named the protuberance a balancer. Their experiments have nothing to do with induction of a balancer. Induction of a balancer is not due to the experimental manipulation as shown by the fact that injection of goldfish DNA or mRNA never produced a balancer-like structure. 5. The use of the electophoretic pattern of lactate dehydrogenase (LDH) to detect organ (liver) specificity. Answer: Liver is one of the few organs possessing LDH-c which migrates toward the cathode. The liver from the hybrid of goldfish and carp is characterized by the presence of 2 bands in between goldfish's 2 bands. Carp liver mRNA injected goldfish were found to have 1 faint band, thus showing the effect of mRNA on the faint band formation. Prof. Davidson considered it unconvincing because the resolution of the bands in the photographs was poor. However, some photographs in the same paper shows the LDH bands from the rat liver mRNA injected fish sharp and clear. STATEMENT BY DR. NIU 100

6. Indirect demonstration of the presence organ (tissue and cell) forming mRNA in the stored mRNA of the egg. Answer: The egg mRNA used in the 1973 experiment has to be tested (as demanded by Prof. Davidson in his writings) for its ability of protein translation. Successful experiments were done with the egg mRNA primed synthesis of albumin and globin. This was the first report suggesting that albumin and globin messages were present in the eggs. Here is what Prof. Davidson wrote, "The presence of albumin or globin message in the egg, even if true, has no particular significance." On the contrary, we believe that if it were true, we would have demonstrated the mRNA nature of the "morphogen" in the egg. The role of the injected rabbit globin mRNA as a "morphogen'' was shown by the appearance of rabbit globin in the red blood cells of the goldfish. 7. Transfer of information from soybean mRNA to rice. Answer: It was reported that soybean protein could be induced in rice by injection of soy seedling mRNA into rice ovaries. Professor Davidson insists that soy globulin is confined to the cotyledons. The mRNA we used was from seedlings with cotyledon removed. Therefore, he claims that the seeds from the injected rice could not possibly contain soy globulin. We have tested the seedling mRNA primed synthesis of soy globulin and the result was positive. Abstract of M.C. Niu's Scientific Research in Academia Sinica, Beijing, China Carp (Cyprino carpio L.) and crucian (Carassius auretus L.) are two species belonging to two genera of the same family, Cyprinidae. They possess a simple forked tail. Goldfish is a mutant of the crucian with double 2-lobed tail. The mating of goldfish and carp or crucian produces offspring with a simple forked tail (i.e., simple tail is dominant and the double tail is recessive). mRNA was prepared from crucian eggs and DNA from liver. Both were injected into goldfish eggs. They develop into fish with simple tails (1). This effect of the egg-mRNA was unexpected but was confirmed repeatedly in the following two years by careful analysis of the fish developed from goldfish eggs injected with carp egg mRNA (2). It should be noted here that the goldfish egg-mRNA injected carp or crucian eggs did not change the simple forked tail into a double fancy type (3). The injection of rat liver-mRNA and rabbit globin- mRNA into goldfish eggs resulted in the development of goldfish with a liver that had some rat liver property (4) and red blood cells with rabbit RBC properties (5). The effect of foreign DNA injected into eggs was also repeated by the injection of California newt (Taricha torosa L.)-DNA into goldfish eggs. Approximately 1 percent of the injected fish developed a newt larval organ, the balancer. This balancer was characterized by three criteria: (1) position, (2) histological structures and the time of appearance and resorption (6,7). STATEMENT BY DR. NIU 101

The goldfish with a simple forked tail induced by nucleic acids were mated among themselves. About one-third to one-quarter of the offspring possess the simple forked tail, thus showing the transmission of the character, induced simple forked tail, to the next generation (8). These findings lead us to explore the nature of the effect of the cytoplasm on the nucleus in Xenopus. White Xenopus is a mutant of the wild type (black). The pigment development from composite eggs (white nucleus in black cytoplasm) is close to the wild type. After metamorphosis, the pigment is confined to the dorsocaudad region (9). Subsequently, we studied the mechanism by which egg-mRNA affect development. We found that following injection of mRNA, DNA synthesis occurred in the egg cytoplasm. We then showed that the egg cytoplasm contained a reverse transcriptase (10). This suggests that this reverse transcriptase catalyzes the transcription of the mRNA into DNA. We then showed that H3-thymidine labeled DNA injected into the egg cytoplasm is transferred to the egg chromosomes (11). Based on these data we suggested that cDNAs transcribed from the cytoplasmic mRNAs are transferred into egg chromosomes and maintained in the chromosomes during development. Our experiments showed that DNA extracted from the fish developed from eggs injected with rabbit reticulocyte mRNA contains the complimentary sequence of the rabbit globin cloned cDNA (11). However, this rabbit globin DNA, though present in other tissues of the fish, is expressed only in the red blood cells. References 1. Tung, T.C. and M.C. Niu (1973). Scientia Sinica, 16:377-384. 2. Tung, T.C. and M.C. Niu (1977). Scientia Sinica, 20:59-63. 3. Niu, M.C. (1981). In ''Role of RNA in Development and Reproduction", Eds. M.C. Niu and X.H. Zhuang. Science Press (Beijing) and Van Nostrand Reinhold, New York, Cincinnati, Toronto, London and Melbourne, pp. 415-433. 4. Niu, M.C. and T.C. Tung (1977). Scientia Sinica, 20:803-806. 5. Niu, L.C., Xue, G.X. and M.C. Niu (1981). In "Role of RNA in Development and Reproduction", pp. 407-414. 6. Tung, T.C. and M.C. Niu (1977). Scientia Sinica, 20:56-58. 7. Tung, T.C. and M.C. Niu (1983). Scientia Sinica, 26:803-806. 8. Tung, T.C. and M.C. Niu (1975). Scientia Sinica, 18:225-228. 9. Yu, J.K., C.P. Shi, and M.C. Niu (1987). Scientia Sinica, 30:487-494. 10. Beljanski, M., Niu, L.C., Beljanski, M.S., Yan, S.Y. and Niu, M.C. (1988). Cell. Mol. Biol. 34:17-25. 11. Niu, M.C., Xue, G.X., Niu, L.C. and H.Z. Huang (1989). Cell. Mol. Biol. 35:333-345. STATEMENT BY DR. NIU 102

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