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E Statement by Dr. Niu OBSERVATION ON THE EVALUATION OF DR. NIU'S RESEARCH AT IDE PUBLISHED IN '`BIOTECHNOLOGY IN CHINA" BY DRS. HAMER AND KI]NG There are several points in Drs. Hones and Kung's evalumbon pages upon which I would like to comment: 1. Based on Hexagonal competition for research grant' IDB got 1.S million yuan from NSFC for 3 years and not for one year. 2. About He working hypothesis (my theory): The stored mRNA in the presence of reverse ~nscnp~se (R~ is uans~ibed into cDNA which is then inserted ink He genome. This cDNA would act as an agent for He differential gene activation during em~yogen~s. DTS. Hamer and Klmg wrote '4This theory directly contradicts He basic laws of Mendelian inheritance." As a developmental biologist, I associate fully what T.H. Morgen wrote ~ his book entitled Embryology anal Genetics that "during cleavage heterogeneous cytoplasm modifies nuclear func- tion." The question often being awed is what is Be component (agent) of the heterogeneous cymplamn? An the seventies we proposed that He egg mRNA transept (cDNA) was the agent. Now in He later half of He 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 reps on Be phenomenon. 3. leaner and Kung stated that He rabbit globin mRNA used in our experi- ment was impure. This was wrong and it strongly indicates Hat dopey did not reed the 97

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98 APPENDIX E paper. In Me abstract of We article we Ranked Prof. Jerry Lingrel for He gift of rabbit globin mRNA (it was He best available at the lime, 1978-1988). In order to show Hat red blood cells (RBC3 from the rabbit globin mRNA injected few carry ~ properties of rabbit RBC, we chose to Erect rabbit globin from ~e Ashes RBC by immuno-precipitation Highly specifics and by He tissue specific lactate de- hydrogenase DASH). The elec~horetic pauem of He liver homogmams from ~ injected fee was of the hybrid type (i.e., intermediate between goldfish and rabbit). Therefore, for our purpose, Here was no need to do globin chemistry as demanded by Homer and Kung. 4. Their comments about the `'green fish" were totally invent No such comments were made. Drs. Namer and Kung ldbelled me as a poor professor without funding, unable to attract students ~ posters. In view of my redre- ment, Drs. Hamer and Kung should know university nobles governing reared professors. As a master 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 alist Of my publications In Chinese and Westem journals, get to How He content and evaluate Pose papers published while worldling in IDB (1982-1990) and not only Pose from Institute of Zoology (1973-1981~. lDrs. Homer and Kung did not know Hat He late Prof. Tung suggested ~ 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 He past few years. The evaluation was supposed to be on papers published by members of IDB. Drs. Homer and Kung chose to comment only papers published in Be period of 1973-1981. Their error was in not e~raluadng the we done at He IDB during He period 1982 -1990; however, Drs. Homer and Kung did mite a sentence about Dr. Niu~s work on cytoplasmic DNA synthesis and the discovery of an independent reverse ban~rip~se in He mid- l980's. Dr. Niu does express great appreciation to DT. Is Ris, a well-known biology gist, for his leper of s~port and cndcism therein of He Drs. Homer and Kung's false statements. Drs. Homer and Kung wrongfully drew the conclusion Cat Dr. Niu's group controls He institute's resources, positions, and power. Therefore, Be research conducted by this group is open to question. Dr.. Niu's scientific and professional response to these irle~ons~le state- ments was an offer already made for He impart selection of a highly res~ted, qualified member of the world scientific community to come to Be Institute at Beijing. All expenses would be paid and He scientist would have He permission of He IDB ~ monitor and see He results of the research conducted by Dr. Niu and to white an independent report of his findings. The offer by Dr. Niu was refused. Obviously, the writers are Prehensive of finding a failure to corroborate their

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APPENDIX E RESPONSE TO PROF. DAVIDSON'S COMMENTS ON M.C. NIU'S SCIENTIFIC RESEARCH IN ACADEMIA SINICA, BEIJING, CHINA 99 Prof. Davidson subscribes to He dogma that bansfannation in developing systems is based on DNA and never on RN~ He also accepts He oncog~c effect of certain veal RNA based on the presence of He reverse rinse (RT for short) in He virus. In eggs, he argues, RT has not been demons Prof. Davidson, in 1989-1990, was not familiar win He work of Niu at IDB after 1982 and presents an exct from his manuscript wntten in 1981. There are seven points in Pro Davidson's analysis of Niu's work in collabo ration with the late Pro T.C. Tung at the Astute of Zoology in Beijing. I shall answer them point by point. 1. The memos of our isolation of mRNA is Reliable because of He use of "Sigmacell" lope 38 cellulose. Answer. New methodology in research develops year after year. By 1972 there were 3 new techniques in He literate for He isolation of mRN~ (1) Absorption to millipore (see I ~~ et al., PNAS, 68:1331, 1971 and Ros~feld et al., B. B. Res. Commun., 47:787, lg72), (2) mRNA hybndi~ion to the oligo AT cellulose (Aviv et al., PNAS, 69:1406, 1972), and (3) isoLddon of eukaryodc mRNA on cellulose and its transLadon in Duo (Schultz et al., B. B. Res. Commun., 16:377, 1972~. The method of using ~Sigmacell" cellulose for mRNA isolation is simple and easy. I he mRNA isolated with this technique was shown ~ be pure and could pie the translation of a Cc protein. The reason we chose "Sigm~ell" cellulose was mainly economical. It goes without saying that in Philadelphia we used "sigma- cell" cellulose first and then purified He mRNA further by oligo ~ cellulose. The LANA acidity was tested by in vitro synthesis of a specific protein. 2. The mRNA and DNA mediated mil transformation Tom He double 2- lobed tad! to He ford ~1 simple goldfish tail is questionable. (Correction: 100 o.d~ml DNA is misprint of 10 o.d~ml) Answer: The claim of nucleic acid-mediamd genetic change was sensational in early 1970. Many biologists were Skeptics because Hey claimed Cat the strain of He goldfish we used was not genetically pure. Ano~erendcism was that we did not use any controls. However we had used fish liver mRNA, rRNA, and minor mRNA as controls and eggs injected win these mRNAs developed into fish without changed tails. They did not accept Cat mRNA could produce genetic change. This is to say Hat He scientific community demands Hat we repm He elements. We decided to use mRNA from a different Species. When egg mRNA Tom a carp, Cypanus carpio L. was injected into goldfish eggs of a pme strain maintained by inking ~ Be Institute of Zoology in Beijing, He tail was changed but win slightly reduced frequency. (Ligature says that cloned rabbit globin gene (Ca 600 b.p.) and many other genes win less than 1,000 b.p. can mediate genetic change.)

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100 APPENDIX E 3. The ~sformation of the mRNA and DNA ~ndluced goldfish tail to next generation. Answer: Published observadon ~4ah~i,J. Imp. Fishenes Instimte, 30:1, 1934) and our own data show that Me tail of the offspring mom the moss of goldfish and crucial carp ar carp is simple and never double 2-lobed. The double 2-lobed tail is recessive. The injection of carp mRNA into goldfish eggs result in He alteration of Be tail fin, while injection of goldfish egg mRNA into carp eggs provided no change of tail fin. These data show the specificity of He carp egg mRNA in goldfish tail change. The making of the simple tailed goldfish obtained Tom the injected eggs Aced offspring win simple forked and double 2-lobed tails, Pus showing the Dan~nission of He induced simple tail to He next generation. This refutes Prof. Davidson assumption Hat 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 He balancer in goldfish larvae. Answer. Balancers are transitional organs found in He California newt, Gotcha torosa. In newt larvae, Hey emerge 5~ days afw ferdlizabon and resorb cat 25 days later. They consist of a pair of bar-like organs on He site post-lateral to Be mouth. In sections they consist of epidermis and connective tissue. The cells at Be top are secretory in nature. When Tancha torosa DNA was injected into goldfish eggs, approximately 1 percent of the larvae developed one balancer on the right or left side. If DNA mom the Chinese news Cynops orientalis, was injected into goldfish eggs, 6 percent of He larvae developed balanced. Over 90 percent of He inject eggs developed nonnally. Shi et aL (Scienda Sinica, 24:402~06, 1980) repeated this expedient using DNA isolated from bog and Chinese newt Over 90 percent of their injected eggs developed abnormally . Among Be abnonnal tadpoles, 0.9 x 1~30f He flog DNA and O.3 x 1~2 percent of the newt DNA-inject developed lateral protuberance on the Speculum. Without considenng the definition of the newt's balances, they named the protuberance a balancer. Their expenmen~ have nothing to do with induction of a balances. 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 ~ balancer-like structure. 5. The use of He electophoredc pattern of lactate dehydrogenase (LDH) to Duct organ (liver) ppecifici~r. Answer: Liver is one of the few organs possessing LDH-c which migrates toward He cathode. The liver mom the hybrid of golf fish and cop is ch~actenzed by ~epresenceof2bands In between goldfishes 2 bands. Carp liver mRNA injected goldfish were found to have 1 faint band, Bus showing the effect of mRNA on the faint band connation. Prof. Davidson considered it unconvincing because the resolution of He bands in Be photographs was poor. However, some photographs in He same paper shows Be LDH bands from the rat liver mRNA injected fish sharp and clear.

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APPENDIX E 101 6. Infect demonstration of He hence organ (tissue and cell) forming mRNA in he stomi mRNA of he egg. Andover: The egg mRNA used in the 1973 experiment has ~ be tested (as demanded by Prof. Davidson in his wridngs) for its ability of protein translation. Successful experiments were done win he egg mRNA primed synthesis of aIbunun and globin. This was the first Sport suggesting Cat albumin and globin messages were present in he eggs. Here Is what Prod Davidson wrote, The presence of albumin or globin message In He egg, even if true, has no particular si~f~cance." On He conuary,we believe that if it were true, we would have demons~a~ He mRNA name of the"morphogen" in the egg. The role of the injected rabbit globin mRNA as a "morphogen" was shown by He appearance of rabbit global in the ~ blood cells of the goldfish 7. Transfer of information Mom soybean mRNA to nce. Answer. It was reported Hat soybean protein could be induced in rice by injection of soy smdling mRNA into rice ovanes. Professor Davidson insists Hat soy globulin is conSned to the cotyledons. The mRNA we used was Tom stings And cotyledon removed Therefore, he claims Cat the seeds Tom die injected rice could not possibly contain soy globulin. We have tested He sting mRNA pled synthesis of soy globulin and Be result was positive. ABSTRACT OF M.C. NIU'S SCIENTIFIC RESEARCH IN ACADEMIA SINICA, BEIJING, CHINA Carp (Cypano carpio L.) and crucian (Carassius auretus L.) are two species belonging to two genera of the same family, Cyprinidae. They possess a simple fold tall Goldfish is a mutant ofthecrucian with double2-lobedtail.The mating of goldfish and carp or cn~cian produces off~pnng with a simple finked mil (i.e., simple tail is dominant and the double tail is recessive3. mRNA was prepared from crucian eggs and DNA from liver. Both were injected into goldfish eggs. They develop into fish win simple tails (1~. His effect of He egg-mRNA was unexpected but was confirmed r~pea~y in the following two years by careful analysis of Be fish developed from goldfish eggs injected with carp egg rnRNA (2~. It should be noted here that the goldfish egg-mRNA injected carp or crucian eggs did not change the simple forked ~1 into a double fancy type (3~. The injection of rat liver-mRNA and rabbit glob~n-mRNA into goldfish eggs resulted in the development of goldfish uric a liver Hat had some rat liver proper (4) and red blow cells with rabbit RBC properties (5~. The effect of foreign DNA injected into eggs was also repeated by He injection of California newt (Tancha 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 msorption (6,7~.

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102 APPENDIX E The goldfish wig a simple forked tail induced by nucleic acids were maid among themselves. About one-~ird to one~uar~ of the offspring possess He simple forked Ail, Bus showing He transmission of the character, induced simple for~d tail, to He next generation All. These findings lead us to explore the natllIe of the effect of He cytoplasm on the nuclei n Xenopus. Whdie Xenopus is a mutant of He wild type (black). The pigment development fiom composite eggs (white nucleus in black cytoplasm) is close to He wild type. After metamorphosis, the pigment is confined to He dorsal caudad region (9). Subsequently, we studied He mechanism by which egg-mRNA affect devel- opment. We found that following injection of mRNA, DNA synthesis occurred In the egg cytoplasm. We then showed thee He egg cytoplasm contained a reverse nanscrip~se (103. This suggests Cat this reverse nan~riptase analyzes the nan~iphon of He mRNA into DNA. We Men showed Eat H3-~ymidine Helen DNA injected into He egg cytoplasm is nansfelr" ~ Be egg chromosomes (11~. Based on these data we suggested that cDNAs Uans~ibed mom Be cympl~c mRNAs are ~sferred into egg chromosomes and maintained in Be chromosomes during development 0~ experiments showed Nat DNA emcee from Be fish developed Tom eggs injected win rabbit reticulocyte mRNA contains Be compli- mentary sequence of the rabbit globin cloned cDNA (11~. However, this rabbit globin DNA, Cough present in other tissues of the fish, is expressed only in the red blood cells. REF E:RENCES 1. T=g, T.C. Id M.C. Niu (1~3~. Science Shim 16:3~-3~. 2. Tung' T.C. and M:C. Niu (1977~. Scientia Sinica, 20:59~3. 3. Nip M.C. (19811. ~ "Role of RNA in Development and Reproduction", Eds. M:C. Niu and X.H Zhuang. Science Pass (Beijing3 and Van Nostrand Reinhold, New Yolk, Cincinnati, Toronm, London and Melbourne, pp. 415433. 4. Niu, M.C. and T.C. Tung (1977~. Scientia Sinica, 20:803-806. 5. Niu, L.C., Xue, GX. and M.C. Niu (1981~. In Whole of RNA in Development and Reproducdon", pp. 407414. 6. Tung, T.C. and M.C. Niu (1977~. Scientia Sinica, 20:5~58. 7. Tung, T.C. and M:C. Niu (1983~. Scientia Sinica, 26:803-806. 8. Twig, T.C. and M:C. Niu (1975~. Scientia Sinica, 18:225-228. 9. Yu, J.K, C.P. Shi, and M:C. Niu (19873. Scientia Sinica, 30:487~94. 10. Bel~aniki, M., Mu, L.C., Belianski, M:S., Yan, S.Y. and Niu, M:C. (19881. CeLL Mol. Biol. 34:17-25. 11. Niu, MiC., Xue, GX., Niu, L.C. and HZ. Huang (1989~. CelL Mol. Biol. 35:333-345.