National Academies Press: OpenBook

Biotechnology in China (1989)

Chapter: 7. Research Highlights

« Previous: 6. Biotechnology Literature Survey
Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"7. Research Highlights." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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7Research Highlights During the course of the literature survey, it became apparent that 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 reports on individual research institutes, in Chapter 8. X-RAY CRYSTALLOGRAPHY X-ray crystallography is the method of choice for determining the precise three-dimensional structure of many biologically important 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 Institute of Physics, Fan Haifu and colleagues have been working 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 initio random-start phasing techniques. The advantage of such methods is that phasing can be determined without the need for multiple measurements at different wavelengths on heavy atom derivatives. Recently, Fan's group has demonstrated the accuracy of their methodology by redetermining the structure of avian pancreatic peptide from one-wavelength, anomalous-scattering x-ray data at 2 Å resolution. Ultimately, this methodology may make it RESEARCH HIGHLIGHTS 32

possible to determine the structure of a variety of peptides and proteins by direct means. This would have broad and important implications for protein engineering. Groups at the Beijing Institute of Biophysics and the Fujian Institute of Research on the Structure of Matter have used more traditional diffraction methods to solve the structures of several interesting molecules such as insulin and trichosanthin. A comparison of native and despentapeptide insulin at 1.5 Å resolution revealed important differences in the position of the B-chain aminoterminal region. The positions of 84 water oxygen atoms and some hydrogen bonds have also been determined. These observations provide important clues to the design of new insulin molecules with altered receptor- activation and multimerization properties. The structural determination of trichosanthin, an abortifacient from a traditional Chinese medicine, revealed it to be a two-domain protein. The three-dimensional structure revealed striking homology to the ricin A-chain, a potent toxin, immediately suggesting the mechanism of action. It was recently reported that trichosanthin selectively kills cells infected with the human immunodeficiency virus, which causes the acquired immune deficiency syndrome (AIDS). Such findings put this research in an interesting new light. REFERENCES Pan Kezhen, Lin Yujuan, Fu Zhuji, Zhou Kangjing, Cai Zhaoping, Chen Zhiwei, Zang Yongmao, Dong Yicheng, Chen Shizhi, Wang Jiahuai, Zhang Xuejun, Ni Chaozhou, Zhang Zhiming, Xia Zongxiang, Fan Zhaochang, and Tian Gengyuan, "The Three-Dimensional Structure of Trichosanthin Molecule," Scientia Sinica (B) 30 (1987): 386-394. Dai Jinbi, Luo Meizhen, You Junming, and Liang Dongcai, "Refinement of the Structure of Despentapeptide (B26-30) Insulin at 1.5 Å Resolution, Scientia Sinica (B) 30 (1987): 55-65. Qian Jinzi, Fan Haifu, and Gu Yuanxin, "Combining Direct Methods with Isomorphous Replacement or Anomalous Scattering Data V. Comparison of Different Phasing Methods in OAS Case," Acta Crystallographica (A) 41 (1985): 476-478. CONTROL OF PLANT VIRUSES Plant viruses cause great harm to China's agriculture. Scientists at the Beijing Institute of Microbiology have been using molecular biological techniques to fight two of the most important menaces, cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). CMV 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 the aphid vectors are difficult to control and no CMV-resistant host genes have been discovered. In RESEARCH HIGHLIGHTS 33

1976, an American group found that certain strains of CMV carry a small RNA, termed satellite RNA, in addition to the four major genomic species. Satellite RNA, which is completely dependent on its helper virus for replication, bears no nucleotide sequence homology to CMV but can interfere with its replication and ability to cause disease symptoms. Tien Po's group at the institute has been studying various strategies to use satellite RNA as a biological control agent for CMV. They prepared two attenuated strains of CMV by coinfecting plants with genomic RNA from Chinese tomato or green pepper together with satellite RNA from a laboratory strain. Greenhouse experiments showed that inoculation of green pepper plants with the attenuated green pepper strain reduced infection by virulent CMV by up to 75 percent over a 10- to 20-day time frame. Plants inoculated less than 10 or more than 30 days prior to challenge were less well protected. An obvious risk to this approach is that the attenuated strain might actually be virulent in other species, thus greatly reducing the practicability 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 strain did not accelerate infection by other plant viruses or pathogens. Indeed, infection of tobacco appears to actually inhibit the growth of certain fungi such as Phytophthora infestans and Cladosporium fulrum. It is presumed, but not proven, that this fortuitous resistance is caused by an interferon-like response to the infection. Additionally, in some cases, it is claimed that infection with the attenuated strains can accelerate plant growth and increase fruit yields. Between 1981 and 1985, the attenuated viruses were field tested on green pepper and tomato plants in several localities throughout China. A 5-year test on green pepper plants in Beijing, Handan, and Yantai gave 70 to 80 percent decreases in disease indexes and 20 to 55 percent increases in fruit 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 fruit yield. Since 1986, these control agents have been applied to much larger areas constituting about 3 percent of the 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 the attenuated virus approach has had success, it would obviously be preferable to develop plants that are permanently resistant to viral infection, and, consequently, bypass the 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 the Ti plasmid, and obtained transgenic tobacco. Most of the transformed plants produce large amounts of satellite RNA and are 10-fold more resistant to CMV infection than are control or nonsatellite RNA-producing plants. The transgenic plants grow almost as well as normal plants both in the greenhouse and in the field; the influence on yield and quality is now being tested. An interesting difference from Baulcombe's results is RESEARCH HIGHLIGHTS 34

that these plants are resistant in inoculated as well as systemically infected leaves. This might reflect a difference between the replication of the monomer satellite cDNA insert used by Tien as compared with the dimer insert used by Baulcombe. Mang Keqiang's group is using a related, but distinct approach to combat TMV. It has long been known that inoculation of tobacco with a mild strain of virus confers resistance to later superinfection with a more virulent strain, a phenomenon known as cross-protection. In 1986, Abel and colleagues showed that such cross-protection could be conferred by the expression of a single protein, the TMV coat protein, in transgenic plants. Following this lead, Mang's group cloned the coat protein gene from the common Chinese strain of TMV and confirmed its identity by DNA sequencing and comparison with the vulgar strain 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 transgenic tobacco plants. Such plants have been shown to produce the coat gene RNA and protein. Although they are still susceptible to TMV infection, symptoms are delayed by 1 to 2 months, which is a substantial advantage under field conditions. The basic mechanism(s) 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 that could form the basis for a useful Sino-American collaborative research project. American scientists may also be interested to know that field testing of genetically altered organisms is possible in China. Given the 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 counterparts. 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, X.F., Wang, K.L., Fang, R.X., and Mang, K.Q., "The Transfer of Chimeric TMV- Coat 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 that there are 100 million antibody-positive individuals in China, many of whom are at RESEARCH HIGHLIGHTS 35

high risk for chronic liver disease and hepatocarcinoma. A notable characteristic of HBV is that it can be transmitted before or at birth from infected mother to baby. A vaccine, derived from the serum of HBV carriers, has been available in the West and in China for several years. However, this vaccine is very expensive (more than $100 in the United States), thus prohibiting the mass inoculations needed to eradicate the disease. Moreover, no matter how well the vaccine material is purified, there is always the fear that it might be contaminated with unknown viruses. Thus, there is a strong incentive to use biotechnology to produce a safe and inexpensive HBV 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, have been particularly active in this field. China is pursuing two strategies to develop such a vaccine. The first, already in use in the United States and Europe, is to employ hepatitis B surface antigen (HBsAg) as a subunit vaccine. Toward this end, the strain of HBV most prevalent in China was cloned and characterized by complete DNA sequencing. The HBsAg-coding sequences were then expressed by using several different eukaryotic vector systems: yeast plasmids with high-efficiency promoters; bovine papilloma virus vectors that can stably replicate in mouse cells; dihydrofolate reductase (DHFR) vectors that can be amplified in Chinese hamster ovary (CHO) cells; and vaccinia virus. In agreement with previous findings of Western scientists, HBsAg was shown to be appropriately glycosylated, assembled into particles, and secreted into the medium of the cultured mammalian cells. The most efficient systems have been transferred to the Biological Products Factory at Changchun (Jilin Province) for medium- scale production. Phase one human trials on two 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 trials of the two Chinese products that will compare them with a vaccine produced in Shenzhen using foreign recombinant DNA technology and with the Chinese serum vaccine. After determining which preparation gives the best protection against HBV infection, large-scale production will be started at Changchun. Attempts to develop second-generation HBsAg subunit vaccines are also under way. One strategy is to produce a mixture of the short form of HBsAg together with a longer form containing the pre-S2 region. The rationale is that both forms are found in serum particles and that the longer variant may contain a binding site for albumin; theoretically, this might increase antigenicity, but clinical trials will be required to test the actual efficacy of this approach. A second idea is to produce synthetic peptide 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 HBV in China: the high cost, the necessity for multiple injections, and the requirement for careful storage of the vaccines. With these problems in mind, RESEARCH HIGHLIGHTS 36

the Beijing Institute of Virology is attempting to develop a more practical vaccine based on live vaccinia virus recombinants. Toward this end, the HBsAg gene was linked to a strong viral promoter and then inserted into the Tian Tan strain of vaccinia virus; this is a vaccine strain that 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 that testing of this material on humans would begin in 1988, but apparently there was some controversy because the recombinant virus was originally plaqued on an established human cell line, and therefore, might have "picked up" some "tumor 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 virus and herpesvirus. There are three noteworthy points about China's efforts to develop new HBV vaccines. First, there has been real cooperation between basic science units (CAS and the Chinese Academy of Preventive Medicine) and a downstream production facility. Second, China is making its own comparison of various forms of the HBV vaccine rather than 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 HBV problem with a novel approach—the vaccinia virus recombinants—which probably will not be clinically tested in the West. References Gan Renban, Chu Meijin, Shen Luping, Qian Suwen, and Li Zaiping, "The Complete Nucleotide Sequence of the Cloned DNA of Hepatitis B Virus Subtype adr pADR-1," Scientia Sinica (B) 1 (1986): 55-65. Wang Yuan, Feng Fongming, Wu Xue, Chen Zhenzhen, Zhong Wuwei, Wu Xiangfu, Kong Yuying, Dai Peihua, and Li Zaiping, "The Recombinant Vaccinia Virus Containing Hepatitis B Surface Antigen Gene—A Potential Live Vaccinia against Hepatitis B," Scientia Sinica (B) (1986): 623-634. Shen Luping, Wong Erli, Pan Tiecheng, Dai Peihua, Li Zaiping, and Li Yuyang, "The Expression of HBsAg Gene in Yeast under Gal-10 Promoter Control," Scientia Sinica (B) 29 (1986): 856-863. Xu Laigen, Hu Peisheng, Chen Zhenzhen, and Huang Weide, "Synthesis and Immunogenicity of Unconjugated Polypeptides of Hepatitis B Surface Antigen," Acta Biochimica et Biophysica Sinica 18 (1986): 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): 4832-4834. RESEARCH HIGHLIGHTS 37

Tsao, H., Ren G.F., and Chu, C.M., "Gene Coding for the Late 11,000-Dalton Polypeptide of the Tian Tan Strain of Vaccinia Virus and Its 5'-Flanking Region: Nucleotide Sequence," Journal of Virology 57 (1986): 693-696. Hou, Y.T., Yang, X.K., and Hu, Y.W., "Variation in the Hind III Restriction Fragments of DNA from the Chinese Tian Tan Strain 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 from cells with desirable traits introduced by conventional genetic selections or recombinant DNA methods; rapid propagation of valuable flowering and fruit-bearing plants; eradication of certain persistent viral diseases; and generation of haploid species with useful properties. Although the principles of plant cell and tissue culture are straightforward, the actual application can involve testing hundreds or thousands of variations in culture conditions. Chinese scientists have achieved several notable accomplishments both in culturing new species and in applying this technology commercially. Chinese scientists were among the first to succeed in the use of anther culture to breed haploid wheat and sugar cane varieties, 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. Meristem 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 the 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 the Japanese) are just beginning to exploit, is that valuable flower varieties can be exported without the usual plant pest control restrictions. In the field of plant regeneration, Chinese scientists have succeeded in obtaining maize embryonic calli from pollen and protoplasts, soybean from protoplasts, and rice (Indica 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 transferring such genes to useful plant species. References He Dinggang and Ouyang Junwen, "Callus and Plantlet Formation from Cultured Wheat Anthers at Different Developmental Stages," Plant Science Letters 33 (1983): 71-79. RESEARCH HIGHLIGHTS 38

Ouyang, J.W., He, D.G., Feng, G.H., and Jia, S.E, "The Response of Anther Culture to Culture Temperature Varies with Growth Conditions of Anther-Down Plants, Plant Science 49 (1987): 145-148. Guo Hongfeng and Ouyang Junwen, "The 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 Zhang Lanying, "Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L. (potato)," Acta Botanica Sinica 30 (1988): 21-24. Li Gengguang and Zhang Lanying, "Regeneration of Fertile Plants from Cotyledon Protoplasts in Solanum melongena L. (eggplant)," Acta Genetica Sinica 15 (1988): 181-184. RESEARCH HIGHLIGHTS 39

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