National Academies Press: OpenBook

Biotechnology in China (1989)

Chapter: Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA

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Suggested Citation:"Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 92
Suggested Citation:"Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 93
Suggested Citation:"Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 94
Suggested Citation:"Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 95
Suggested Citation:"Appendix D: An Analysis of Niu Menchang's Research on Transformation by RNA." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Page 96

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DAn Analysis of Niu Menchang's Research on Transformation by RNA Eric H. Davidson It has been claimed by Niu and his associates that amphibian and teleost eggs can be transformed heritably with injected egg poly(A) RNA.27-32 Because of the potentially great practical, experimental, and theoretical importance of embryo germ line transformation, it is worth examining these claims carefully. In the following paragraphs, a brief review of the published reports of Niu et al. is presented. Unfortunately, this review leads to the conclusion that these claims of RNA-mediated embryo transformation are incorrect. To obtain germ line transformation, the introduced sequences must, of course, be incorporated into the DNA genome. It could be argued that retroviral reverse transcriptase is present in eggs (though there is absolutely no evidence of this), and could copy the injected maternal poly(A) RNA into DNA that could then be incorporated into the genome. Were this likely to occur, however, it would follow that there should also be a high frequency of reverse transcription and reinsertion of the endogenous maternal poly(A) RNA. Current evidence, on the contrary, proves that this type of event is very rare, and that it occurs only on an evolutionary time scale. Several pseudogenes have recently been found33-36 that indeed could have appeared in the germ line by the route of reverse transcription and insertion. The distinguishing feature of such genes is that they have the structure of mature Eric H. Davidson is the Norman Chandler Professor of Cell Biology at the California Institute of Technology, Pasadena, California, and he is a NAS member. NOTE: This analysis is an excerpt from a paper, ''The Maternal RNA of Amphibian and Echinoderm Eggs,'' which was submitted for publication in Scientia Sinica in 1982. It and an earlier Letter to the Editors were never published. AN ANALYSIS OF NIU MENCHANG'S RESEARCH ON TRANSFORMATION BY RNA 92

messages rather than of genes. That is, they lack the intervening sequences possessed by most genes but they have terminal poly(A) sequences that in functional genes are not coded in the genomic DNA. Direct sequence analysis often displays sequence changes in the pseudogenes that have required millions of years to accumulate. This, plus the facts that only a few if any such pseudogenes are ever found for any given true gene and that most genes are single-copy sequences, shows that incorporation into germ line DNA of sequences copies from mRNA is an exceedingly infrequent event. It is consequently impossible to believe a priori that RNA injected into an egg could be efficiently reverse transcribed and incorporated in the genome. Otherwise, the genome would contain enormous numbers of copies of genes without intervening sequences, while, of course, exactly the reverse is true. In addition, the traits claimed to have been transformed by Niu and his associates are in general morphological, and their developmental creation almost certainly requires the participation of many genes working together. To transform such traits would necessitate incorporation of all the requisite genes in intact form, plus their control systems, which is even more unlikely. Biological principles aside, the reports of Niu et al. are experimentally unconvincing. Specific examples of probable artifacts, misinterpretations, or inadequate procedures include the following: 1. The mRNA utilized by Tung and Niu27-29 was extracted by an unreliable method, utilizing a material called "Sigmacell." This is merely an unpurified cellulose, some batches of which trap some poly(A) RNAs. There is, in fact, no evidence that the RNA extracted was polyadenylated, or that it was mRNA. The cell-free translation studies reported by Niu et al.30 were done with RNAs prepared by a better procedure, and these experiments are not relevant to the claim that the RNA in the original reports was mRNA. Furthermore, the gel electrophoresis patterns shown for the alleged mRNA in the 197729 and 197327 papers are impossible for real poly(A) mRNA, which, of course, does not band tightly in one place as illustrated because it is extremely heterodisperse in size. 2. The same transformation results were reported to result from DNA and mRNA injection.27 I am certain the DNA results are an error or at best not the result of nucleic acid injection. Whichever is the explanation, it no doubt pertains to the mRNA results as well because it is even more unlikely that there are two different explanations for the same unlikely observation. The data shown state the DNA was injected at "100 OD/ml," indicating that the DNA was concentrated to about 4,000 µg/ml. Unfortunately, DNA cannot be concentrated to this extent unless it is broken to very small pieces, a few hundred nucleotides long (or is supercoiled, which is irrelevant for this case). DNA a few hundred nucleotides long, of course, cannot transform anything since a single gene is usually at least a few kilobases (kb) and sometimes as much as 20 or 50 kb in length. The proposition that a whole organ-level structure can be transferred heritably by small pieces of DNA is particularly difficult to believe. AN ANALYSIS OF NIU MENCHANG'S RESEARCH ON TRANSFORMATION BY RNA 93

3. In the fish transformation experiments it was claimed that a heterospecific tail phenotype could be induced by mRNA injection. However, the fact that this "single tail" phenotype occurs at a significant frequency, even though breeders "have always discarded those with single fins," may be significant. Tung and Niu27 make the key assumption that single tail must be a ''genotypic character." However, no evidence is presented that single tail is in fact a simple genetic character. In fact, Tung and Niu28 report ratios of single tail to double tail in the single parent offspring of the alleged transformed fish that cannot easily be interpreted in terms of a Mendelian character. I would guess that single tail is a minor developmental abnormality, perhaps an easily induced phenocopy of a complex genetic mutation. Many such abnormalities are known in developmental biology. I suspect this physiological lesion has nothing whatsoever to do with the introduced nucleic acids and is the result of handling, the injection procedure, or some other experimental artifact (see below). 4. Tung and Niu31 also claimed nucleic acid-mediated transfer of an urodele amphibian embryonic trait; the balancer A version of this experiment was recently performed by Shi et al.,37 who showed that, indeed, injection of urodele DNA into an anuran (frog) egg produces an extra "balancer" (i.e., protrusion on the chin of the embryo which may or may not be a real balancer), but so does injection of the DNA of this anuran into its own egg. Therefore, this case is clearly just what I suspect the "single tail" result is, viz., a developmental lesion that can occur naturally, and is simply a frequent experimental artifact stimulated by the operation. It might be noted that this is not the only example of such artifacts. For example, Scharf and Gerhart38 have recently shown that the appearance of extra heads and nerve chords in amphibian eggs can be stimulated merely by turning the eggs over. Previous workers (e.g., see Curtis39 ) had claimed that transfer of cytoplasmic cortical material produced such secondary axiations, but to carry out the experimental transfers the eggs had to be turned on their sides. Scharf and Gerhart38 showed that in fact the material transferred had nothing whatsoever to do with the result of the operation. 5. Niu and Tung32 also claimed that injection of carp mRNA into goldfish eggs produces adult liver isozymes similar to those occurring in carp × goldfish hybrids produced by artificial insemination. The evidence is a series of lactate dehydrogenase (LDH) starch gels. These gels are unconvincing since they are severely overloaded or have been run with such poor resolution that one band cannot be distinguished from another. (For contrast, observe the precise appearance of the LDH isozyme gels in Wright and Subtelny40 ). The key observation purports to show an additional faint band, but the appearance of this band suggests an artifact caused by a salt front in the gel. There is no likely way that a variant could be present at 1/100 the concentration of all the other variants anyway, since all the liver cells must have the same genes, as they all descend from the same allegedly transformed egg. 6. Niu et al.30 claimed that poly(A) RNA of carp eggs can be translated to produce albumin. The procedures in this paper for extraction of RNA are AN ANALYSIS OF NIU MENCHANG'S RESEARCH ON TRANSFORMATION BY RNA 94

acceptable, but everything depends on the specificity of the albumin antibody. No evidence on this point is presented whatsoever. In any case, the claim is irrelevant to the nucleic acid transformation claimed in the earlier papers because the RNA is prepared differently and because the presence of the albumin message in the egg, even if true, has no particular significance. Albumin or some other cross-reactive protein could be used in the early embryo and be coded by a maternal message like so many other proteins. In another RNA transformation study, Niu et al.41 claimed that soybean protein can be induced in rice by injection of soy seedling mRNA into rice ovaries. The RNA was extracted from seedlings from which the cotyledons had been removed. However, J. Bonner (personal communication) has shown that soy globulin is confined to the cotyledons. Therefore, Niu et al.41 injected mRNA from the part of the plant that contains no soy globulin mRNA. Thus, it is impossible that the injected RNA caused the rice cells to synthesize soy globulin. It can be concluded that there is no experimental support for the contention that maternal poly(A) RNA can be utilized for transformation of eggs, nor, on theoretical grounds, should this be possible. It is obvious that the correct route to development of a germ line transformation system is introduction of the genetic material, that is, DNA, not RNA. This is clear from successes already obtained with tissue culture cells,42 Dictyostelium,43 and Drosophila embryos.2526 Embryo DNA transformation will provide a decisive approach to many problems in the molecular biology of early development, including the role of maternal poly(A) RNA. Notes 27. Tung, T.C., and Niu, M.C., Scientia Sinica 16 (1973): 377-384. 28. Tung, T.C., and Niu, M.C., Scientia Sinica 18 (1975): 223-228. 29. Tung, T.C., and Niu, M.C., Scientia Sinica 20 (1977): 59-63. 30. Niu, M.C., Yu, J.K., and Song, D.X., Scientia Sinica 23 (1980): 510-516. 31. Tung, T.C., and Niu, M.C., Scientia Sinica 20 (1977): 56-58. 32. Niu, M.C., and Tung, T.C., Scientia Sinica 20 (1977): 803-806. 33. Hollis, G.F., Hieter, P.A., McBride, O.W., Swan, D., and Leder, P., Nature 296 (1982): 321-325. 34. Reilly, J.G., Ogden, R., and Rossi, J.J., Nature 300 (1982): 287-289. 35. Wilde, C.D., Crowther, C.E., Cripe, T.P., Gwo-Shu Lee, M., and Cowan, J.J., Nature 297 (1982): 83-84. 36. Leder, A., Swan, D., Ruddle, F., Eustachio, P., and Leder, P., Nature 293 (1981): 196-200. 37. Shi, L., Yan, Y.C., Zhang, J.Z., Mo, H.Y., and Lu, Y.Q., Scientia Sinica 24 (1981): 402-406. 38. Scharf, S.R., and Gerhart, J.C., Developmental Biology 79 (1980): 181-198. AN ANALYSIS OF NIU MENCHANG'S RESEARCH ON TRANSFORMATION BY RNA 95

39. Curtis, A.S.G., Journal of Embryology and Experimental Morphology 10 (1962): 410-422. 40. Wright, D.A., and Subtelny, S., Developmental Biology 21 (1971): 119-140. 41. Niu, M.C., Chang, P.Y., Lin, Z.P., Ma, C., and Zhang, Y.L., Scientia Sinica 23 (1980): 119-122. 42. Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R., Cell 16 (1979): 777-785. 43. Hirth, K.P., Edwards, C.A., and Firtel, R.A., Proceedings of the National Academy of Sciences USA, in press. AN ANALYSIS OF NIU MENCHANG'S RESEARCH ON TRANSFORMATION BY RNA 96

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