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D An Analysis of Niu Menchang's Research on Transformation by RNA Eric H. Davidson It has In claimed by N;u and his associates Mat amphibian and teleost eggs can be transformed heritably win injected egg poly(A) RNA.27-32 Because of We potentially gmat practical, experimental, and theoretical importance of embryo germ line bansfonnation, it is worm examining these claims carefully. In the following ~a~gr~hs, a brief review of He published reports of Niu et al. is present. Unfortunately, this review leads to Be conclusion ~ these claims of RNA-mediated embryo uansfonnation are inc~ct. To obtain germ line transformation, Be in~duced sequences must, of course, be incorporated into Be DNA genome. It could be argued that retrovi~ reverse nscnptase is present in eggs Though there is absolutely no evidence of dlis), and could copy tile injected maternal poly(A) RNA into DNA ~ could Ben be incorporated into the genome. Were this likely to occur, however, it would follow Rat Here should also be a high frequency of reverse eanscriphon and reinsertion of He endogenous maternal poly(A) RN~ Current evidence, on He contrary? proves ~ this type of event is very rue, and bat it occurs only on an evoludonaIy dine scale. Several pseudogenes have recently been foamd~36 pal indeed could have appeal in Me genn line by the route of reverse Ascription and insertion. The distinguishing feature of such genes is Hat they have Be structure of mawe Eric H. Davidson is Tic Nonn" Candler Professor of Call Biology at die Ifs Rate of Technology, Misadd, ~lifomia, and he is a NAS member. NOTE: This analysis is an e receipt from a paper, "line lvIatemal RNA of AmEh~ian and Edhinodenn Eggs," which was subjoined far publication m Scientia sinica ~ 1982. It and an eadier ~tar to the Editors were never published. 92

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APPENDIX D 93 messages rather than of genes. That is, Hey lack the intervening sequences possessed by most genes but they have enninal poly(A) sequences Cat in functional genes are not coded in Be genomic DNA. Direct sequence analysis often displays sequence changes in He pseudogenes that have required millions of years ~ accumulate. This, plus He facts that only a few if any such pseudogenes are ever for for any given true gene and that most genes are single~copy sequences, shows Hat incorp~adon into g=II line DNA of sequences copies Mom mRNA is an exceedingly infrequent event. It is consequently unposslble to believe a proton Eat RNA injected into an egg could be efficiently reverse transcribed and plural in the genome. Otherwise, He genome would contain enormous numbers of copies of genes without intervening sequences, while, of course' exactly the reverse is true. In addition, the Nits claimed to have been transformed by Niu and his associate are in general morphological, and Heir developmental caption almost c~inly requires He participation of many genes wowing together. To transform such traits would necessitate incorporation of all He requisite genes in intact fonn, plus Heir control systems, which is even more Likely. Biological principles aside, the reports of Niu at al. are experimentally unconvincing. Specific examples of probable artifacts, misint~pretadons, or inadeq~ procedures include the following: 1. The mRNA Quiz by Tung and NiuZ7-29 was exuacted by an unreliable melody utilizing a material called "Sigmacell." This is merely an unpurified cellulose, some batches of which trap some poly(A) RNAs. There ash in fact, no evidence Hat He RNA extracted was polyedenylated, or that it was mRNA. I he celling uansl~ion studies reported by Niu et alp were done win RNAs prepared by a better procedure, and these experiments are not relevant to the claim Hat the RNA in the original reports was mRN~ Furthermore, the gel electrophoresis Ens shown for He alleged mRNA in Be 1977" and 197327 papas are impossible for real poly(A) mRNA, which, of courm, does not band tightly in one place as illustrated because it is ex~nely heterodipperse in Rind,. 2. The same transformation results were reported ~ result from DNA and mRNA injection.Z7 I am certain He DNA results are an error ~ at best not the result of nucleic acid injection. Whichever is the explanadon, it no doubt per ains to the mRNA results as well because it is even more unlikely that Here are two different explanations for He same unlikely observation. The data shown state the DNA was injected at"100 OD/rnl," indicating Bat He DNA was concentrated to about 4,000 ~ghnt Unfortunately, DNA cannot be concert to this extent unless it is broken to very small pieces, a few hundred nucleotides long (or is s~rcoiled, which is irrelevant for this amp. DNA a few hwKlred nucleoddes long' of course, cannot transf~n anything since a single gene is usually at least a few kilobases fob) and sometimes as much as 20 or 50 kb in length. The proposition that a whole argan-level structure can be transferred heritably by small pieces of DNA is particularly difficult to believe.

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94 APPENDIX D 3. In the fish cansfonnadon experiments it was claimed that a heterospecific tail phenotype could be induced by mRNA inaction. However, He fact Hat this "single tail" phenotype occurs at a significant frequency, even Cough breeders "have always discarded those win single fins," may be significant. Tung and NiP make He key assumption that single tail must be a 4`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 Angle parent offspring of the alleged transformed fish that cannot easily be interpreted in terms of a Mendelian character. I would guess that Angle tail Is a for 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 nosing whatsoever to do win the induced nucleic acids and is the result of handling, the injection pressure, or some other experimental artifact (see below). 4. Tung and Niu3~ also claimed nucleic acid-m~dint~1 transfer of an urodele amphibian embryonic twit; He balancer A version of this experiment was recently perfonned by Shi et al.,37 who showed ~at, indeed, injection of ukelele DNA ink an anuran (~g) egg produces an extra "balances" (i.e., profusion on He chin of He embryo which may or may not be a real balanced, but so aloes injection of the ONA of this anuran into its own egg. Therefore, this case is clearly just what I suspect the "single mil" result is, viz., a development lesion Cat can occur naturally, and is simply a fiequent experimental artifact stimulated by He operation. It might be noted Hat this is not the only example of such artifacts. For example, Scharf and Gerhardt have recently shown that the appearance of extra heads and nerve chords in amphibian eggs can be stimulated merely by turning He eggs over. Previous wooers (e.g., see Curds39) had claimed Hat transfer of cytoplasmic cortical material produced such secondary aviations, but to caky out the experimental transfers the eggs had ~ be toned on Heir sides. Scharf and Gerhart38 showed Cat in fact He material ~arusferred had nothing whatsoever to do win He result of the operation. 5. Niu and Tung32 also claimed that injection of carp mRNA into goldfish eggs produces adult liver Rezones similar to those occ~g in carp x 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 Hat one band cannot tee distinguished Tom another. Q:orcon~ast observe He precise appearance of the LDH isozyme gels in Wright and Subteloy~. The key observation purports to show an additional faint band, but the appearance of this band suggests an artifact caused by a salt Font in He gel. There is no likely way that a variant could be present at l/lOO the concentration of all the other variants anyway, since all He liver cells must have the same genes, as they all descend hum 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

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APPENDIX D 95 acceptable, but evoking depends on He specificity of He albumin antibody. No evidence on this point is presented whatsoever. Ln any case, He claim is irrelevant to He nucleic acid t~fm''~ation claimed in the earlier peers because He RNA is prepared differently and Cause Be presence of the albumin message in ~e egg, even if true, has no par6C',tar significance. Albumin or some over cross- reactive protein could be used in the early embryo and be coded by a matinal message like so many over proteins. ~ another RNA Dansfo~ion study, Niu et al ~ claimed that soybean protein can be induced in rice by injection of soy seedling mRNA into rice ovaries. The RNA was exit Mom Rings mom which He cotyledons had been removed However, J. BoMer (personal communication) has shown Cat soy globulin is canned to the cob,riedons. Therefore, Niu et aim injected mRNA mom the part of He plant that contains no soy globulin mRNA. Thus, it Is impossible that the injected RNA caused He rice cells to synthesize soy globulin. It can be concluded Mat there is no experimental s~port for the contention Hat n~1 poly(A) RNA can be u1ili~ for mason of eggs, nor, on ~eoredcal grounds, should this be possible. It is obvious that He correct route to development Of a genn line transformadon system is introduction of He genetic material, Mat is, DNA, not RNA. Ibis is clear from successes already obtained win tissue culture cells,42 Dic~yostelium,43 and Drosophila embryos. Embryo DNA bansf~nation will provide a decisive approach to many problems in Be molecular biology of early development, including the role of maternal poly(A) RNA. NOTES 27. Tung, T.C., and Niu, M.C., Scientia Silica 16 (1973~: 377-384. 28. Tung, T.C., and Niu, M.C., Scientia Siruca 18 (1975): 223-228. 29. Tung, T.C., end Niu, M.C., Scientia Silica 20 (1977~: 59~3. 30. Niu, MiC., Yu, Joy, and Song, D.X., Scientia Sinica 23 (1980~: 51~516. 31. Tung, T.C., and Niu, M.C., Scientu: Silica 20 (1977): 56 58. 32. Niu, M.C., and Tung, T.C., Scientia Silica 20 (1977): 803-806. 33. Hollis, GO., Hieter, P^., McBride, O.W., Swan, D., and Leder, P., Nature 296 (1982~: 321-325. 34. Reilly, J.G., Ogden, R., and Rossi, JJ., Nature 300 (1982): 287-289. 35. Wilde, CD., Crowther, CE., Cripe, TV., Gw~Shu Lee, he, and Cowan, JJ., Nature 297 (1982): 83-84. 36. Leder, At, Swan, D., Ruddle, F., Eus~hio, P., and Leder, P., Manure 293 (1981): 19~200. 37. Shi, L., Yan, Y.C., ~hang, ~ 7., Mo, H.Y., and Lu, Y.Q., Scientia Sinica 24 (1981): 402406. 38. Scharf, SO., and Gerhart, J.C., Developmental Biology 79 (1980): 181- 198.

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96 APPENDIX D 39. Curtis, A.S.G., Journal of Embryology and E~erimerual Morphology 10 (1962~: 410 4ZZ. 40. Wright, D^, and Subtetny, S., Developmental Biology 21 (1971~: 119- 140. 41. Niu, M.C., Chang, P.Y., Lin, ZP., Ma, C., and Zhang, Yip., Scientza Silica 23~1980~: 119-122. 42. Wigler, Mi, Sweet, R., Sim, G.K., Wold, B., Pelticer, A., Lacy, E., Mdniatis, T., Silverstein, S., and Axel, R. Cell 16 (1979~: 777-785. 43. Each, KP., Edwards, C.A., and Fillet, R.A., Proceedings of the National Academy of Sciences USA, in press.