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7 Research Highlights During tile course of the literature survey, it fine apparent Hat certain research in China has progressed to an international level. The following section describes the most promising of these projects in both basic and applied biotechnology. This material is elaborated on, and in some cases overlaps the report on individual research institutes, in Chapter S. X-RAY CRYSTALLOGRAPHY X-ray crystallography is He method of choice for determining the precise Free dimensional structure of many biologically impart molecules. X-ray structure determination techniques have evolved in two major directions: direct methods, for small molecules; and indirect methods, involving multiple derivative sets of diffraction data, for macromolecules such as proteins. China possesses a high level of expertise in both of these state-of-the-art technologies. At the Beijing Instate of Physics, Fan Hbifu and colleagues have been wowing on probability phasing methods to determine the crystal structures of increasingly large biological molecules. They were among the first to develop and successfully apply ab Patio random-start phasing techniques. The advantage of such methods is that phasing can be determined without He need for multiple measurements at different wavelengths on heavy atom derivatives. Recently, Fan's group has demonstrated the accuracy of their methodology by redetermining He structure of avian pancreatic peptide from one-wavelength, anomalous- scattenng x-ray data at 2 A resolution. Ultimately, this methodology may make it 32

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RESEARCH HIGHLIGHTS 33 possible to determine He structure of a variety of peptides and proteins by direct means. This would have bed end important implications for protein engineering. Groups at the Beijing Institute of Biophysics and the Fujian Institute of Research on Be Structure of Matter have used more traditional diffraction mesons to solve the structures of several interesting molecules such as insulin and ~ichosan~in. A comparison of nadve and despentapepdde insulin at 1.5 A resolution revealed important differences in Be position of the B-chain attune Conical region. The positions of 84 water oxygen amms and some hydrogen bonds have also been determined. These observations provide important clues to Be design of new insulin molecules with altered mceptor-activation and multimerization properties. The struck determination of trichosan~in, an abortifacient from a traditional Chinese medicine, revealed it to be a tw~domain protein. Lee three dimensional structure revealed stri~ng homology to Be ncin A-chain, a potent toxin, immediately suggesting the mechanism of action. It was recently reported Rat ~ichosan~in selectively kills cells infected with the human ~unodeD~ciency virus, which causes the acquired immune deficiency syndrome (AIDS). Such findings put this research in an interesting new light References Pan Kethen, Lin Yujuan, Fu Zhuji, Zhou Kayoing, Cal Zhaoping, Chen Zhiwei, Bong Yongmao, Dong Yicheng, Chen Shizhi, Wang Jiahuai, Zhang Xuejun, Ni Chaozhou, Ehang Zhiming, Xua Zongxiang, Fan Zhaochang, and Tian Gengyuan, 'e The Dimensional Structure of Tnchosanthin Molecule," Scientia Silica ha) 30 (1987): 38~394. Dai Jinbi Luo Mei~hen, You Junming, and Liang Dongcai, '~eSnement of the Structure of Despentapepiide (B2~30) Insulin at I.5 A Resolution, Scientia sauca A) 30 (1987): 55~5. Qian Jinn, Fan Haifu, and Gu Yuanxin, ``Combining Dent Methods with Isomorphous RepLacement or Anomalous Scattering Data V. Comparison of Different Phasing Methods in GAS Case," Acta Crystallographica (A) 41 (1985~: 476478. CONTROL OF PLANT VIRUSES Plant viruses cause great harm to China's agncul~. Scientists at He Beijing Institute of Microbiology have been using molecular biological techniques to fight two of the most important menaces, cucumber mosaic vinus (CMV) and tobacco mosaic vims KIEV). CHIN can infect more than 775 plant species, including green peppers, tomatoes, and tobacco (but, despite the name of the virus, not Chinese cucumbers). Previously, there were no means to prevent viral infection since He aphid vectors are difficult to control and no CMV-resistant host genes have been discovered. Id

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34 BIOTECHNOLOGY IN CHINA 1976, an American group found that cmain swains of CMV carry a small RNA, ~ satellite RNA, in addition to He four major genomic species. Satellite RNA, which is completely dependent on its helper virus for replication, bears no nucleot~de sequence homology to CMV but can interfere with its replication and ability to cause disease symptoms. Tien Po's group at Be institute has been studying various strategies to use satellite RNA as a biological control agent for CMV. They prepared two a~enua~d swains of CMV by coinfecting plants win genomic RNA from Chinese tomato or green pepper together with satellite RNA from a laboratory strain. Greenhouse experiments showed Mat inoculation of green pepper plants with Me attenuated green pepper swain reduced infection by virulent CMV by up to 75 percent over a 1~ to 20 day time frame. Plants inocuLded less than 10 or more than 30 days prior to challenge were less well protect An obvious rick to this approach is that the attenuated swain might actually be virulent in over species, thus gready Educing He pracdcabili~ of field applications. However, infection of 38 different plant species produced no obvious disease symptoms except for slight mosaic symptoms in Cucurbita pepo. In addition, the attenuated CMV swain did not accelerate infection by over plant viruses or pathogens. Indeed, infection of tobacco appears to acmaDy inhibit He growth of certain fun"' such as Phytophthora irfestans and C0osponwn fuirum. It is presumed, but not proven, that this fortuitous resistance is caused by an interferon-like response to He infection. Additionally, in some cases, it is claimed that infection with He attenuated swains can ac&eleram plant grown and increase fruit yields. Between 1981 and 1985, He ~tenuamd viruses were field tested on green pepper and tomato pants in several localities throughout China A 5-year test on green pepper plants in Beijing, Handan, and Yantai gave 70 to 80 percent decmases in disease indexes and 20 to 55 percent increases in Wit yields. A 3-year test on tomato plants in Taiyuan gave a 31 to 40 percent reduction in disease and a 31 to 45 percent increase in Quit yield. Since 1986, these control agents have been applied to much larger areas constituting about 3 percent of He fields around some large cities (e.g., Beijing and Shanghai) and up to 80 percent of the fields around certain small cities (e.g., Handan and Anda). While He attenuated virus approach has had success it would obviously be preferable to develop plants that are permanently resistant to viral infection, and, consequently, bypass He necessity for seasonal inoculations. In 1986, Baulcombe showed that a dimer insert of satellite RNA introduced into tobacco on a tumor- inducing (Ti) plasmid vector could be expressed into biologically active RNA. Following up on this lead, Tien's group obtained and sequenced their own satellite cDNA clone, inserted a monomer into He Ti plasmid, and obtained transgenic tobacco. Most of the transformed plants produce large amounts of satellite RNA and are Refold more resistant to CMV infection than are control or nonsatellite RNA-producing plants. The transgenic plants grow almost as well as nonnal plants bow in the greenhouse and in the field; He influence on yield and quality is now being ms~ An interesting difference Mom Baulcombe~s results is

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RESEARCH lIIGHLIGHTS 35 Hat these plants are resistant in inoculated as well as systemically infected leaves. This might reflect a difference between Be replication of the monomer satellite cDNA insert used by Tien as compared with the dimer insert used by Baulcombe. Mung Keqiang's gropp is using a related, but disduct approach to combat TMV. It has long been known Mat inoculation of tobacco with a mild shin of virus confers resistance to later s~erinfection with a more virulent strain, a phenomenon known as cross-protectiom In 1986, Abel and colleagues showed Cat such cross-protection could be conferred by the expression of a single protein, Be TMV coat protein, in ~nsgenic punts. Following this lead, Mang's group cloned the coat protein gene from Be common Chinese strain of TMV and confined its identity by DNA sequencing and comparison with Be vulgar swain sequence already published by Western scientists. They then fused the coat protein gene to a strong promoter sequence, mobilized the fusion gene into the Ti plasmid, and obtained asnsgenic tobacco plants. Such plants have been shown to produce the cow gene RNA and protein. Although Hey are still susceptible to TMV infection, symptoms are delayed by 1 to 2 months, which is a substantial advantage under field conditions. The basic mechanisms by which expression of satellite RNA or coat protein blocks viral infection is still unknown. Possibilities include competition for replication or packaging factors, blockage of receptors, or a more general response to infection and stress. This is an interesting problem Cat could form the basis for a useful Sino-American collaborative research project American scientists may also be interested ~ know that field testing of genetically altered organisms is possible in China Given He large area of cultivated land and the variety of soil and climate conditions in China, this might provide the basis for joint ventures between American agricultural biotechnology companies and their Chinese countess. References Tien Po, Zhang Xiuhua, Qiu Bingsheng, Qin Bingyi, and Wu Gusui, "Satellite RNA for the Control of Plant Diseases Caused by Cucumber Mosaic Virus," Annals of Applied Biology 111 (1987~: 143-152. Tian, Y.C., Qing, Ad., Wang, Kit-., Fang, RX., and Mang, K.Q., ' The Transfer of Chirnenc TMV(oat Protein Gene and its Expression in Transgenic Tobacco Plant," Proceedings of the Eighth International Conference on Global Impacts of Applied Microbiology and International Conference on Applied Biology and Biotechnology (1988, in press). TOWARD A HEPATITIS B VIRUS VACCINE Hepatitis B virus (HBV), the causative agent of serum hepatitis, is a serious health problem in China, as it is throughout much of Asia. It is estimated Hat Here are 100 million antibody-positive individuals in China, many of whom are at

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36 BIOTECHNOWGYIN CHINA high rick for chronic liver disease and hepatocarcinoma A notable charactenshc of HBV is that it can be bansmi~d before or at bird from infected mother to baby. A vaccine, derived from He serum of HBV comers, has been available in He West and in China for several years. However, this vaccine Is very expensive (more than $100 in the United S~s), thus prohibiting He mass inoculations needed to eradicate He dismse. Moreover, no matter how well the vaccine material is punned, there is always He fear Bat it might be contaminated with unlmown wruses. Thus, there is a strong incentive ~ use biotechnology to produce a safe and inexpensive BV vaccine. The groups of Li Zaiping, at the Shanghai Institute of Biochemistry, and of Hou Yunde and C. M. Chu, both at the Beijing Institute of Virology, leave been particularly active in this field. China is pursuing two strategies to develop such a vaccine. The first, already in use in He United States and Europe, is to employ hepatitis B surface antigen (BsAg) as a subunit vaccine. Toward this end, the strain of BV most prevalent in China was cloned and characterized by complete DNA sequencing. The HBsAg~oding sequences were Men expressed by using several different eukalyotic vector systems: yeast plasmids with high-eff~c~ency promoters; bovine papilloma virus vectors that can stably replicate in mouse cells; dihydrofolate reductase (DHFR) vectors Cat can be simplified in Chinese hamster ovary (CHO) cells; and vaccinua virus. In agreement with previous findings of Western scientists, BsAg was shown to be appropriately glycosyla~d, assembled into Uncles, and secreted into the medium of the cultured mammalian cells. The most efficient systems have been transferred to the Biological Products Factory at Changchun Qilin Provinces for medium-scale production. Phase one human dials on Nero of these products have given encouraging results: no obvious side reactions and a high level of antigenicity. The plan for 1989 is to carry out phase two Dials of the Ho Chinese products Hat will compare Hem with a vaccine produced in Shenzhen using foreign recombinant DNA technology and with the Chinese serum vaccine. AM determining which preparation gives He best protection against BV infection, large-scale production will be sty at Changchun. Attempts to develop second-generation BsAg sublmit vaccines are also under way. One strategy is to produce a mixture of He short form of BsAg together with a longer form containing the pre-S2regiom The rationale is that both forms are found in serum particles and Hat the longer vanant may contain a binding site for albumin; theoretically, this might increase antigenicity, but clinical Dials will be reqali~d to test He actual efficacy of this approach. A second idea is to produce synthetic peptize antigens. Although this research has been publicized in the Chinese press, it seems unlikely to be practical in view of the many studies showing that a much smaller fraction of the human population will respond to a single epitope than to the mixed epitopes of complete HBsAg. While the efficacy of HBsAg subunit vaccines is now established, there remain serious difficulties in using this approach for the massive inoculations needed to eradicate REV in China: He high cost, He necessity for multiple injections, and He requirement for careful storage of the vaccines. With these problems in mind,

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RESEARCH HIGHl]GHTS 37 He Beijing Institute of Virology is attempting to develop a more practical vaccine based on live vaccinia virus recombinants. Toward this end, He HBsAg gene was linked to a strong vim1 promoter and then inserted into the T'an Tan swain of vaccinia virus; this is a vaccine strain Hat has been used extensively for the eradication of smallpox in China. The recombinant viruses have been propagated in primary cells and shown to be antigenic in experimental animals. (For further details and comparison with American research, see Chapter 8.) It was originally planned Hat testing of this material on humans would begin in 198S, but apparently there was some controversy because He recombinant virus was originally plagued On an established human cell line, and therefore, might have "picked ~" some 'mod material." Fortunately, material that has been grown only on primary cells will soon be available for testing. This vaccine has many potential advantages: low cost, stability of the vaccine at room temperature, and single inoculation with an air gun. Ultimately, it may be possible to make multivalent vaccinia viruses that include antigens for hepatitis A vims and herpesvirus. There are Free noteworthy points about Finals efforts ~ develop new HBV vaccines. First, Here has been Hal cooperation between basic science units (CAS and He Chinese Academy of Preventive Medicines and a downs~arn production facility. Second, China is making its own comparison of venous forms of the BV vaccine miner Han simply accepting Western findings; this is critical in view of the different target groups (children and potential mothers in China versus doctors, dentists, and sexually active male homosexuals in the United States). Lastly, China appears ready to respond to the urgency of its HElV problem with a novel approach~the vaccinia virus recombinant~which probably will not be clinically tested in to West. References Can Renban, Chu Meijin, S hen Luping, Qian Suwen, and Li Zaiping, 'the Complete Nucleodde Sequence of He Cloned DNA of Hepatitis B Virus Subtype adr pAOR-l," Scientia Sinica ~) 1 (1986~: 55-65. Wang Yuan, Feng Fongming, Wu Xue, Chen Zhenzhen, Zhong Wuwei, Wu Xiangfi~, Kong Yu~g, Dai Peihua, and Li Zaiping, `-The Recombinant Vaccine Vines Containing Hepatitis B Surface Antigen Gen~APotendal Live Vaccinia against Hepatitis B." Scientza Sznzca I) (1986): 623~34. Shen Luping, Wang Erli, Pan Tiecheng, Dai Peihua, Li Zipping, and Li Yuyang, '`The Expression of HBsAg Gene in Yeast under Gal-10 Promoter Control," Sczentia Sznica (B) 29 (19863: 85~863. Xu Laigen, Hu Peisheng, Chen Zhenzhen, and Huang Weide, "Synthesis and Immunogenicity of Unconjuga~Polypep~des of Hepatitis B Surface Antigen," Acta Bzachimzca et Biophyszca Sznzca l 8 ~19861: 513-519. Tsao, H., Liu, G.Q., Ruan, L., and Chu, C.M., "Construction and Application of Plasmids Containing Bidirectional Promoters of Vaccinia Virus," Journal of Virology 62 (1988): 4832~834.

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38 BIOTECHNOLOGY [N CHINA Tsao, H., Ren G.F.' and Chu, CM., "Gene Coding for He Late 11,000-Dalton Polypep~de of He Tian Tan Sin of Vacua Virus and Its S'-Hanking Region: Nucleodde Sequence," Journal of Virology 57 (1986~: 693-696. Hou, Y.T., Yang, X.K., and Hu, Y.W., '~Variabon in He Hind m Rest:riction Fragments of DNA Tom the Chinese Tian Tan Spain of Vaccinia Virus," Journal of General Virology 66 (1985): 1819-1823. PLANT CELL AND TISSUE CULTURE The in vitro culture of plant cells and tissues has numerous applications in agricultural biotechnology: the regeneration of plants Tom cells win desirable waits induced by conventional genetic selecdons or recombinant DNA methods; rapid propagation of valuable flowering and ~uit-beanng plants; eradication of chin persistent viral diseases; and generation of haploid species with useful properties. Although He principles of plant cell and tissue culture are straightforward, He actual application can involve testing hundreds or thousands of variations in culture conditions. Chinese scientists have achieved several noble accomplishments both in culturing new species and in applying this technology commercially. Chinese scientists were among Be first to succeed in He use of anther culture to breed haploid wheat and sugar cane vaneties, many of which are now in widespread field use. For examples of the painstaking research used to optimize culture conditions, see the references below by Ouyang Junwen and colleagues. Menstem culture has been used to obtain plants free of two important viral pests, the potato degeneration virus and gladiolus mosaic virus. Tissue culture is routinely used in several Guangdong institutes for He rapid propagation of various fruiting and flowering plants such as grape, orchid, gladiolus, chrysanthemum, African violet, narcissus, and fuchsia An important advantage of this technology, which the Chinese (and He Japanese) are just beginning to exploit, is that valuable flower vmedes can be echoed without the usual plant pest conned resuicdons. In He field of plant regeneration, Chinese scientists have succeeded in obtaining Moe embryonic calli Tom pollen and pro~lasts, soybean from protoplas~cs, and rice (Ind`;ca strain) from embryos and protoplasts. Many Western scientists are now working on the isolation of plant genes of economic importance. Collaboration with Chinese scientists could provide an important step in mnsfernng such genes to useful plant species. References He Dinggang and Ouyang Junwen, `4Callus and Plantlet Formation from Cultured Whew Angers at Different Developmental Stages," Plant Science Letters 33 (1983~: 71-79.

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RESEARCH HIGHUG~S 39 Ouyang, J.W., He, D.G., Feng, G.H., and Jia, SE, 4`The Response of Anther Culture to Culture Temperature Varies win Grown Conditions of Anther- Down Plants, Plant Science 49 (1987): 145-148. Guo Hongfeng and Ouyang Junwen, '4The Effects of KNO3 Concentration in Callus Induction Medium for Wheat Anther Culture," Plant Cell, Tissue and Organ Culture 12 (1988): 3-12. Li Gengguang and rehang Ian~g, "Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L. (po~to),"ActaBotanica Sinica 30 (1988): 21-24. Li Gengguang and Zhang Lanying, '!Regeneradon of Fertile Plants from Cotyledon Protoplasts in Solanum melongena L. (eggplant)," Acta Geneaca Sinica 15 (1988): 181-184.