National Academies Press: OpenBook

Biotechnology in China (1989)

Chapter: 8. Current Research at Selected Institutes

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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Suggested Citation:"8. Current Research at Selected Institutes." National Academy of Sciences. 1989. Biotechnology in China. Washington, DC: The National Academies Press. doi: 10.17226/2074.
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Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

8Current Research at Selected Institutes The following section describes current biotechnology-related research at 19 institutions that were visited during a 1-month evaluation trip to China. The purposes of this somewhat anecdotal recounting are threefold: to support, in a concrete way, the general conclusions of Chapter 3 on how research funding is prioritized and allocated; to elaborate on research trends and quality examined in Chapters 6 and 7; and to provide a road map for American scientists interested in cooperation and collaboration with Chinese colleagues. A list of contacts at these institutions is included in Appendix C. BEIJING Beijing Agricultural University Beijing Agricultural University, formed in 1952 from the agricultural departments of Beijing and Qinghua Universities, is China's largest agricultural university with a staff of 1,050, including 100 professors, 235 associate professors, and 350 lecturers. The large campus on the outskirts of Beijing includes an experimental farm, and the university also operates experimental stations in Hubei and Harbin. Of the 21 departments, five actively pursue research involving biotechnology. The amount and type of equipment in the research laboratories have recently been improved by using funds from a World Bank loan. The university operates a national key laboratory for agricultural biology and plans to establish an open laboratory for agricultural biotechnology in the near future. The generation of transgenic plants is of interest to several groups at the CURRENT RESEARCH AT SELECTED INSTITUTES 40

university. Yun Longfei's group has cloned a soybean 7S storage protein gene and shown, in agreement with results of other groups, that it is expressed in a tissue-specific fashion in Ti plasmid-generated transgenic tomato, potato, and tobacco plants. They are also attempting to generate CMV-resistant plants by using the strategy employed at the Beijing Institute of Microbiology. Attempts to clone plant photosynthetic and high nutritional value protein genes, by using probes from other groups, are still at the genomic library construction stage. Work on transgenic animals at the Beijing Agricultural University focuses on the use of growth hormone and growth hormone-releasing factor genes to improve the growth characteristics of pigs and rabbits. Although the relevant genes have already been cloned by Western biotechnology companies, they are trying to get their own genes and are still in the process of making genomic libraries. Chinese Academy of Agricultural Sciences The Chinese Academy of Agricultural Sciences was established in 1957 to coordinate agricultural research activities at the national level. Currently, it has 33 research institutes, a graduate school, an agricultural library, an agriculture and technology publishing company, a center for computer sciences, and the Center for Biotechnology. There are a total of 10,575 staff members, including 5,063 scientists, 812 administrative personnel, and 4,700 support staff. The Center for Biotechnology was established in 1986 with a mission that includes research and training. According to the original plan, the center will have 40 new positions. At the present time, there are three major laboratories: molecular biology and genetic engineering, plant cell technology, and tumors and monoclonal antibodies. Research subjects carried out at the center include vaccine and genetic engineering, plant genetic engineering, biological control and viral genetic engineering, protoplast culture and fusion, and plant virus and monoclonal antibody studies. The Molecular Biology and Genetic Engineering Laboratory is led by Y.L. Fang, director of the center. Currently, there are about 20 people: one associate professor, two instructors, one postdoctoral fellow, three Ph.D. students, eight M.S. students, and five research associates. This laboratory is well funded with approximately 400,000 yuan from the Chinese government and $20,000 from the Rockefeller Foundation. They are carrying out four major projects. In the isolation and identification of plant genes project, the genes that have been cloned are leghemoglobin and 11S seed storage protein from soybean. In the nutritional improvement of crop species project, they are at the stage of constructing genomic libraries from alfalfa. In the insect-resistant plants project, they are transferring plants with Bacillus thuringiensis toxin gene by using β- glucuronidase as the reporter gene. The isolation of toxin genes from insects project has just been started. The Plant Cell Technology Laboratory is led by S.C. Chia, who is currently CURRENT RESEARCH AT SELECTED INSTITUTES 41

taking sabbatical leave in Singapore. He has a group of nearly 10 people consisting of one associate professor, two research associates, and several M.S. students. This laboratory has been credited with making contributions in fusing cucumber protoplasts and subsequently regenerating them into plants. It receives good support at 200,000 yuan per year. The Tumor and Monoclonal Antibody Laboratory conducts research on potato viruses. It is the newest among the three laboratories. Currently, there are about 10 people receiving about 200,000 yuan per year for research. Institute of Biophysics (CAS) The Beijing Institute of Biophysics, founded in 1959, currently houses some 800 workers, including 400 scientists. The institute is divided into 12 departments that include, in biotechnology-related fields, bioengineering, enzymology, x-ray crystallography, protein engineering, and cell biology. An interview conducted with Lei Kejian concentrated on scientific topics rather than research support. However, it was obvious from the sophisticated instrumentation in the laboratories that the institute is well funded. The research highlight at the institute is their internationally recognized work on structure-function relationships in insulin. These studies involve high- resolution x-ray crystallography to determine the precise structure of the molecule, chemical and enzymatic modifications to determine the roles of various residues in molecule function, and genetic engineering to produce novel derivatives. The structure of native insulin (Zn2 hexamer form) has now been completed at a 1.2 Å resolution, the highest yet reported. At this resolution, it is possible to visualize three hydrogen bonds and to detect asymmetry at two disulfide bonds. The structure of chemically prepared despentapeptide insulin, which lacks five residues and has lost 8 percent of its biological activity, has been solved at a 1.5 Å resolution and appears identical to the native form except for the position of the carboxy-terminal residue. Narrowing in on the active site, attempts are now under way to solve the structure of deshexapeptide insulin, which lacks one additional residue and has lost all biological and receptor- binding activities. Structure-function relationships of insulin are also being studied by a comparative approach, and the structure of a fish liver insulin has been solved at a 2.8 Å resolution. Building on this basic research, the protein engineering group is attempting to engineer long-acting forms of insulin. Insulin is stored and secreted as a Zncoordinated hexamer, whereas the active form is either a monomer or dimer (a point of dispute among the Beijing Institute of Biophysics and other groups). Therefore, if the association constant for the hexamer could be increased, administered insulin might have a longer action time. Using computer graphics, it was shown that there is an empty area between adjacent dimers in the hexamer structure, and that a valine and a glutamic acid residue reside on opposing forces of this region. Molecular orbital calculations suggested that changing the valine CURRENT RESEARCH AT SELECTED INSTITUTES 42

to arginine, a basic amino acid, would allow formation of a salt bridge and substantially increase the association constant. This change has now been achieved by site-directed mutagenesis, and mass production in E. coli is under way. Another useful alteration, which has been achieved by chemical methods, is the deletion of phenylalanine residue B1. This produces an insulin which is still 100 percent biologically active but lacks immunological reactivity, an important step in treating diabetics that mount an immune response to administered insulin. The same change is now being made by genetic engineering methods to allow large-scale production. Two organizational aspects of the insulin work are noteworthy. First, although the bulk of funding is from High Technology Program grants, it is clear that there is a major nonapplied component to this work and that the researchers' hearts are really in basic research. Second, the work has proceeded in close collaboration with groups at the Beijing Institute of Physics, the Shanghai Institute of Biochemistry, and Peking University. Such collaborative research, especially between CAS and non-CAS institutes, is unusual in China; the success of the insulin work illuminates its importance. The Beijing Institute of Biophysics is pursuing research on the structure of several other interesting proteins and small molecules. The structure of trichosanthin, a component of a traditional Chinese medicine used as an abortifacient, has been solved at 2.6 Å (R = 0.29), and data for the 1.8 Å map have been collected. The three-dimensional structure shows an even closer resemblance to the ricin A-chain, a related ribosome inhibitory protein, than does the primary structure. In collaboration with the visual sciences group at the institute, a lightsensing protein has been purified to homogeneity, and crystallization attempts are under way. The structures of several small molecules, such as a natural antimalarial drug, have also been solved. Institute of Developmental Biology (CAS)* When the Beijing Institute of Developmental Biology was established in 1980, it was the realization of the vision of T.C. Tung, who, during his years as a highly respected senior embryologist at the Beijing Institute of Zoology, had inspired the idea of creating a separate institute of embryology or developmental biology. Although Tung died in the late 1970s, his idea was adopted and promoted by Niu Mann-Chiang, a Chinese-American biologist who had worked in Tung's laboratory when he started splitting his time between China and his own laboratory at Temple University. Niu capitalized on his association with Tung, and Tung's relationships with government leaders including Deng Xiaoping, to orchestrate the founding of the institute. * This section has been revised from the earlier edition. For a statement by Dr. Niu regarding this section, see Appendix E. CURRENT RESEARCH AT SELECTED INSTITUTES 43

The institute houses 60 scientists, including 13 senior researchers and 15 research associates. Although it does not have a formal Ph.D. program, there are several doctoral students in Niu's laboratory. Several of the institute's students and scientists have studied abroad, where they have shown themselves to good advantage. The institute is divided into five research groups that are working on 21 individual projects. Current support from the High Technology Program, the Seventh 5-Year Plan, and NSFC is 1.8 million yuan per year, plus an undisclosed amount from CAS specifically for Niu's laboratory. The institute occupies a unique position among China's biology research organizations. Under Niu's leadership, the institute obtained $650,000 from the Rockefeller Foundation, $550,000 from the United Nations Fund for Population Activities, and several million yuan from the Chinese government in order to build and equip its research facility. The institute's laboratories are the most modern, well equipped, and best maintained of those the authors visited in China. Clearly, Niu has been influential in the establishment of this institute, a role that is reflected in his position and the amount of funding the institute has been able to attract. However, the following overview of Niu's research casts doubt on the extent to which his enquiries can be counted as contributions to the international scientific community. The focus of Niu's research is on the role of RNA in biological systems and during development. In 1975, he claimed that the fungus Neurospora crassa could be stably, genetically transformed by RNA.* This is a remarkable claim since it is universally believed that in Neurospora, as in all other organisms, it is DNA rather than RNA that serves as the genetic material. Moreover, despite intense work on the genetic transformation of Neurospora, this observation has never been reproduced in the published literature. Because transformation experiments on Neurospora are easy to perform, it is likely that these experiments have in fact been repeated but without positive results. Niu has also claimed that RNA can be stably inherited in higher organisms and can cause the derepression of normally silent genes. For example, in 1977 he reported that mouse uterus injected with RNA from rats or chicken synthesized both mouse and the donor species albumin in uterine epithelial cells, suggesting that the mouse genes were depressed by the heterologous RNA. However, it is difficult to interpret these experiments because the method used to detect albumin synthesis was not sufficiently documented. Moreover, similar experiments using cloned, purified genes and well established gene transfer methods have failed to reveal any derepression of the endogenous genes. Starting in 1981, Niu described experiments in which goldfish developing from eggs microinjected with rabbit globin mRNA were purported to express rabbit globin, as determined by an immunological assay. The results were again * Mishra, N.C., Niu, M.C, and Tatum, E.L., Proceedings of the National Academy of Sciences USA (1975): 642-645. CURRENT RESEARCH AT SELECTED INSTITUTES 44

ambiguous because the globin was not purified or adequately characterized. Subsequently, successful gene transfer has been reported in fish, but these experiments all used DNA rather than RNA as the nucleic acid and purified genes rather than a crude mixture of species. Judging from the rate of transformation with purified genes, it is difficult to believe the results claimed by Niu using total, unfractionated RNA containing thousands of different messages. The most spectacular of Niu's claims is the ability of RNA to redirect the formation of whole organs in intact animals. Specifically, he claims to have created goldfish that have balancer appendages or tail shapes derived from newts or carp, and that these new traits can be inherited. Such fish, if they existed, would be of intense interest to many biologists. However, when the authors asked to see these animals, Niu claimed that they are all kept at another location even though the Institute of Developmental Biology has extensive fish- raising facilities on its own campus. If and when these fish are made available to other investigators, it should be straightforward to check them for the presence of newt or carp genetic information using modern methods. Only when these tests have been conducted will it be possible to determine whether the fish represent a real phenomenon. Appendix D, provided by Eric Davidson, provides a further analysis of the experimental and theoretical problems raised by Niu's work. The laboratory of Yan Shaoyi, Director of the Institute of Developmental Biology, studies nuclear-cytoplasmic interactions in fish. His group has shown that it is possible to recover fertile adult fish from enucleated eggs injected with the diploid nucleus from early embryonic cells. In agreement with work by other laboratories on amphibians, they observe that the ability to recover viable progeny declines with the developmental stage of the donor cells. They have also made hybrids between different species of fish and claim that it is possible to obtain viable progeny from crosses between different genera and some different subfamilies, but not between different families. While most of the hybrid progeny appear to be identical to the nucleus donor species, they also make the surprising claim that some offspring display morphological traits similar to those of the recipient species or intermediate between those of the donor and recipient. Even more remarkably, they claim that the recipient traits can be passed on to offspring and that the penetration of the recipient characteristics can be increased by serial transplantations. One example, which has been reported widely in the Chinese press, is the cross between nuclei of the common carp Cyprinus carpio L. and enucleated eggs of the crucian carp Carassius auratus L. It is claimed that this hybrid has a higher growth rate, higher protein content, and lower fat content than the common carp and that it can be bred stably. Yan attempts to explain these results by a theory in which ''silent genes'' in the donor nucleus undergo activation and rearrangement under the influence of the recipient cytoplasm. However, a major flaw of these experiments is the lack of distinct markers for the donor nucleus. Without such markers, there is no way to be sure whether the "hybrid" fish are really derived from the nucleus of the donor or from residual material of CURRENT RESEARCH AT SELECTED INSTITUTES 45

the recipient. There are some simple biochemical and molecular biological experiments that could clearly resolve this question, but Yan shows no inclination to perform these analyses. Another serious flaw is the lack of quantitative analysis and controls to show that these fish are not simply mutants induced by the various experimental manipulations. Until these controls are performed, Yan's results must be viewed with skepticism. Xiao Shuxi studies DNA polymerase in Erlich ascites cells. She claims to have discovered a new polymerase with a different pH optimum and size than any of the polymerases in normal cells and hopes to use it as a marker for cancer cells. However, the experiments are impossible to interpret because of a complete lack of controls. Moreover, since the Erlich cell polymerase has not actually been purified, the idea of making diagnostic reagents is premature. The laboratory of Lu Deyu works on the genetic manipulation of mammalian embryos. They have spent several years developing an electroporation method to perform nuclear transplantation in rabbits. The idea is to test Niu's cytoplasmic activation of the nucleus theory in a mammal, but so far no progeny animals have been recovered. They are also working on the production of transgenic farm animals by the more conventional DNA injection method and claim to have obtained one cow which expresses HBsAg. Visits to the laboratories of two associate professors, Wu Naihu and Wu Zhengan, demonstrated that the Institute of Developmental Biology does conduct some research at an international level. Wu Naihu studies the rice chloroplast genome. He is continuing his work, started in Ray Wu's laboratory in the United States, on the structure and regulated expression of various chloroplast genes such as psbA and psbB. He has also made the interesting observation that in certain crosses between different rice species, the F-1 plants display a new chloroplast DNA restriction band. This is quite surprising, since the chloroplast genome is known to be maternally inherited. Wu Zhengan studies highly repeated DNA sequences in amphibians and mammals. He has made the interesting observation that an extra C band in one chromosome of a Chinese ground squirrel is due to the transposition of a satellite DNA. He is also studying a series of satellite DNAs from Chinese newts and has shown that one form is specific to Asian species. Wu's work demonstrates the use of modern molecular techniques to study the basic biology of uniquely Chinese species. He was proud to note that his recent work, which has been published in an international journal, was conducted exclusively in China. In summary, the quality of the research conducted at the Beijing Institute of Developmental Biology is highly variable. The work in several of the laboratories, particularly that of Niu, is so poorly controlled and documented that the results will continue to be questioned in China and abroad. Continued support for such research will not advance developmental biology in China, sets a poor example for other Chinese scientists, and casts doubts on the efficacy of peer review CURRENT RESEARCH AT SELECTED INSTITUTES 46

procedures. On the other hand, the institute does have some laboratories, such as those of Wu Naihu and Wu Zhengan, that are performing creditable research on worthwhile topics. Given China's general weakness in developmental biology (see Chapter 6), it is urgent that this institute focus its considerable resources on supporting the best possible research. Institute of Genetics (CAS) The Beijing Institute of Genetics employs 470 workers, including 80 senior scientists, 180 middle-level scientists, and 40 graduate students. There are 40 research groups working in the areas of molecular genetics, gene and chromosome engineering, plant breeding and tissue culture, animal developmental genetics, and human and medical genetics. Approximately 70 percent of the research is on plants. Funding is obtained through CAS, NSFC, the Seventh 5-Year Plan, the National Key Project Program, and the High Technology Program. Approximately half the research is classified as basic, half as applied. Located in the new Datun Road complex, it maintains an experimental farm and animal facility, as well as a laboratory building. Plant regeneration is a key research area and a point of pride for the Beijing Institute of Genetics. Ouyang Junwen's laboratory was the first to achieve anther culture of wheat and has continued to work on this project for the past 10 years. By systematically varying the medium and temperatures used for anther cultivation and callus induction, they have achieved up to 10 percent plant propagation efficiencies. They have also shown that the efficiency of callus formation and the percentage of albino plantlets, a serious problem in anther culture, are both complex multigenic traits. A focus of current research is the use of anther culture to obtain mutant plants resistant to diseases, such as the wheat scab fungus, Fusarium species, or to herbicides such as atrazine. Two scab-resistant lines have been isolated and are currently being field tested. Attempts are also in progress to obtain wheat with a high lysine content, by selection with a lysine analog, but the investigator did not seem convinced of the physiological soundness of this approach. The laboratory is also beginning gene transfer attempts, using marker genes such as kanamycin resistance, but there was no clear focus on what useful genes might be employed in the future. In testimony to the somewhat unique position of this laboratory in the anther culture field, Monsanto Company has contracted with Ouyang to culture and provide seeds from their germplasm. The laboratory is supported by 140,000 yuan for 5 years from the Seventh 5-Year Plan and High Technology Program grants and $60,000 from Monsanto. Li Xianghu's laboratory concentrates on the culturing, fusion, and DNA transformation of plant protoplasts. Over the past 10 years, this group has successfully regenerated plants from rice, tobacco, petunia, and others and has had initially encouraging results with wheat—a species so far resistant to this technique. They recently made front-page headlines in the Chinese press by CURRENT RESEARCH AT SELECTED INSTITUTES 47

transferring an α-interferon gene into tobacco. The transgenic plants produce low levels of interferon (1,000 units per gram of leaf tissue) and are currently being tested for resistance to TMV. Curiously, Li's group has not done the control experiment of determining whether direct interferon treatment protects against this virus. Zhen Zhu, who recently obtained his Ph.D. from the University of Tennessee and who holds a Rockefeller Foundation fellowship to return to the United States for 3 months each year, is studying the more basic problem of the relationship between methylation and gene expression in plants. Another very interesting basic project is an attempt to create new plant species by protoplast fusion. This group is handsomely supported by 700,000 yuan for 5 years from the High Technology Program and the Seventh 5-Year Plan. Li Liangcai's group also works on DNA transformation of plants using a two-step protoplast preparation method worked out in his laboratory. They have transferred two marker genes, those for kanamycin resistance and β- glucuronidase, in both stable and transient transfection systems. The main emphasis is on methodology rather than potential applications. The laboratory is supported by 150,000 yuan for 5 years from the Seventh 5-Year Plan and High Technology Program and by $15,000 from the Rockefeller Foundation. From the above review, it is clear that the Beijing Institute of Genetics conducts research in several areas of modern biotechnology and gene manipulation. Therefore, it was surprising to note that few—if any—of the senior scientists have a background in classical genetics, and that the institute does little research in the traditional areas that provide the foundation for modern molecular biology. Institute of Microbiology (CAS) The Beijing Institute of Microbiology houses 580 workers, including 236 middle- and senior-level scientists and 20 Ph.D. students. There are eight divisions: mycology, bacteriology, virology, genetics, physiology, ecology, natural resources, and a fermentation facility. The institute is supported by 6 million yuan per year, of which 60 percent is derived from various project grants. The mycology and bacteriology divisions, which are concerned mostly with systematics, have recently been designated as a national key laboratory with two postdoctoral positions. Plant virology is a highlight of the institute's biotechnology research. Tien Po's laboratory studies CMV, which infects several important crops such as tomatoes, green peppers, and tobacco. During propagation, small satellite RNAs that depend on the main genome for their replicative functions occasionally occur. Tien's laboratory and other laboratories showed that these satellite RNAs can interfere with the replication and disease-causing properties of superinfecting viruses. Therefore, they prepared a mixture of CMV and excess satellite particles and showed, in greenhouse experiments, that it protected plants against CMV superinfection. This "plant vaccine" can be administered either by individual inoculation of plants or, in more recent experiments, by a convenient spray gun CURRENT RESEARCH AT SELECTED INSTITUTES 48

technique. Subsequently, large amounts of the material have been prepared in the institute's fermentation facility and have been used on tomato and green pepper plants in several parts of China. Tien claims that infections, in some cases, have been cut by 50 percent and yields increased by 30 percent. Current research focuses on incorporating the interfering gene sequence into the tobacco genome to provide permanent protection. Mang Keqiang, together with young colleagues Qing Xiaofong and Yang Maozhou, work on TMV, a close relative of CMV that also infects tomatoes and tobacco. Building on work performed in the United States, they are attempting to prevent TMV infection by persistent expression of the coat protein of the virus in transgenic plants. The idea is that the coat protein, which normally forms the viral envelope, will bind to cell surface receptors and therefore compete with virus particles for a binding site. This group cloned a Chinese strain of TMV, showed that it has a sequence very similar to that of a previously characterized American strain, and introduced the coat protein gene into tobacco by Ti plasmid transformation. The resulting transgenic plants express coat protein, and although they are still susceptible to TMV infection, the symptoms are delayed by 1 to 2 months under field conditions. Mang's laboratory has also carried out comparative studies of various isolates of CMV, TMV, barley stripe mosaic virus, wheat mosaic virus, cereal mosaic virus, and a gladiolus virus by using DNA sequencing, RNA sequencing, and antibody techniques. The Beijing Institute of Microbiology also has a long history in industrial fermentation. Between 1972 and 1978, they developed a simple two-step fermentation technique for the production of vitamin C, and in 1985, this process was sold to Hoffmann-La Roche, Incorporated. They are currently working on the production of enzyme inhibitors to increase the efficiency of various β-lactam antibiotics against resistant strains of bacteria, and on a kit for blood cholesterol analysis by using two enzymes produced by fermentation. Other fermentation studies involve protoplast fusion of useful penicillin producers and characterization of a new virus in Aspergillus niger. Institute of Virology (CAPM) The Institute of Virology, which is part of the Chinese Academy of Preventive Medicine (CAPM; previously Chinese Academy of Basic Medical Sciences), is the major research center for animal virology in China. Although a substantial proportion of the institute's research is in classical medical virology, there are four projects involving molecular biology: bio-engineered vaccines; basic studies of vaccinia virus replication; production of interferon, interleukin-2, and other lymphokines; and protein engineering. This institute, with 12 High Technology Program grants and 18 Seventh 5-Year Plan grants, is one of the two most richly supported research institutes in China. They are currently erecting a national key laboratory funded by 5 million yuan from the State Planning Commission. CURRENT RESEARCH AT SELECTED INSTITUTES 49

Although the physical facilities appear dilapidated and are located in a hutong, or alley, district far from other institutes in Beijing, a relatively high proportion of scientists and students were seen to be actively performing experiments during several visits. Groups under the supervision of the institute's director Hou Yunde and former director C.M. Chu are involved in several biotechnology projects. One major area of interest is the use of vaccinia virus as a vector for vaccine production. They have constructed a new vector with two important differences from the vectors used by Bernard Moss's laboratory in the United States. First, they use the hemagglutinin gene rather than the thymidine kinase gene as an insertion site because hemagglutinin mutants can be grown on normal cell lines, whereas the thymidine kinase mutants must be propagated on a special cell line. Moreover, the virus yield is higher. Second, the Chinese vector is based on the Tian Tan strain, which has been widely used for smallpox vaccination in China. The construction of the new vector required a substantial amount of basic research on vaccinia virus gene expression, including the sequencing of 30,000 base pairs of the vaccinia virus genome. An unexpected result of this research was the discovery that the vaccine virus hemagglutinin gene is a member of the immunoglobulin gene superfamily. Their results suggest that hemagglutinin binds to lymphocytes in a manner analogous to that of the CD2 surface protein with T lymphocytes. Much of the basic research has been published in Western journals. Development of a vaccine against HBV is another priority of the institute. Closely following work in the West, institute scientists cloned and sequenced the surface antigen gene of a Chinese HBV strain, inserted it into a DHFR vector, and introduced it into CHO cells. After amplification by methotrexate selection, the cells were found to secrete 5 to 7.5 mgl of HBsAg per liter that was appropriately glycosylated and assembled into subviral particles. It is possible to culture the cells, and collect the media every 2 days, for up to 120 days. In animal tests and phase one human clinical trials, the genetically engineered vaccine is four times more potent as an antigen than is the current human plasma vaccine. The vaccine, which meets World Health Organization standards, is currently produced and purified in a small laboratory at the institute. The production process is similar to one developed by Genentech in the United States. Production and genetic engineering of interferon is a long-standing project. Using standard E. coli methods, they have produced interferon which appears to be clinically useful against chronic cervical condylomas caused by human papillomaviruses. They have also produced hybrids between interferon and tumor necrosis factor, which they hope will target the interferon to tumor cells, and between interferon and the pre-S region of HBsAg, which they hope will direct it to liver cells. These experiments also address the basic question of which parts of the interferon molecule are responsible for its antiviral activity. The Institute of Virology's work on viral replication is one of China's few examples of basic research that has garnered notice internationally. At the same CURRENT RESEARCH AT SELECTED INSTITUTES 50

time, its work on vaccine development shows a sensitivity to practical needs as well as to the necessity of tailoring Western inventions to China's needs. In summary, this institute shows that it is possible to integrate basic and applied research in the context of China's new scientific policies. Institute of Zoology (CAS) The Beijing Institute of Zoology is a multidisciplinary research center with an emphasis on animal reproduction and classical fields such as taxonomy and ecology. Biotechnology-related research is carried out in the Laboratory of Genetic Engineering, which is supported by a Seventh 5-Year Plan grant, under the supervision of Shen Xiaozhou. The genetic engineering group focuses on the biology and applications of growth hormones. Using techniques similar to those developed at Genentech and Integrated Genetics Corporations, they have generated a CHO cell line carrying a metallothionein-human growth hormone fusion gene and linked DHFR gene. After methotrexate amplification, the cell line produces and secretes high levels of human growth hormone (100 µg/106 cells/day) which will be used for the treatment of hereditary dwarfism. This group is also trying to create "superfish" by microinjection of a mouse metallothionein-bovine growth hormone fusion gene into freshly fertilized eggs of the common carp. Of 100 injected eggs that developed into fish, 30 contained the foreign gene, as shown by appropriately controlled Southern blots. Of these, 10 displayed rapid growth compared with control fish, and three of these that survived past 1 month are now being bred to test for germline inheritance. The creation of transgenic cows and sheep is also under way. In order to create appropriate vectors, student Yang Weimin has performed interesting and competent basic research on the organization of the metallothionein gene cluster in the cow. Peking University Peking University is China's most famous institution of higher learning both inside and outside the country. Many of China's leaders, including Mao Zedong, have at least passed through, if not been graduated from, this prestigious and historic university. The Department of Biology at Peking University is staffed by a large faculty of 20 full professors, 40 associate professors, and more than 100 teaching assistants and lecturers. The rotating chair of the department is currently held by Gu Xiaocheng, an articulate spokesman for the role of universities in China's research program. The biology department has several subdivisions: botany (including plant physiology, plant genetics, and plant cell biology), zoology (including insect physiology and ecology), cell biology, genetics, biophysics, microbiology, biochemistry (the largest division), and a special teaching group to coordinate and teach courses for the 640 undergraduates in the department. There are 150 M.S. CURRENT RESEARCH AT SELECTED INSTITUTES 51

students and 50 Ph.D. students. Undergraduates are admitted on the basis of a national test whose results are curved to fit the requirement for geographical quotas. The department also provides training for 90 premedical students from the Beijing Union Medical College. Of the 12 students who received Ph.D.'s in 1987, five stayed on as instructors and the rest either went abroad or took faculty positions at other universities. The department is richly supported by some 6 million to 7 million yuan in research grants held in 1988. This represents a 150 percent increase over funding levels in 1987 and a more than 1,000 percent increase over levels in 1983. Of the current grants held, 11 are from the High Technology Program, 5 are from the Seventh 5-Year Plan, and 55 are from NSFC. Nearly 90 percent of the senior faculty hold research grants. The State Education Commission provided the construction costs for the department's new laboratory facility and supplies 300,000 yuan per year for teaching costs, but it provides no direct research support. The rather lavish equipment for the new laboratories was purchased with a loan from the World Bank. In addition, the Department of Biology houses a national open laboratory on protein engineering and has received funds for five national postdoctoral fellow positions. The department's prominence is evidenced by their winning more Natural Science Awards than any other department in China and by having an NSFC approval rating of 50 percent compared with 30 percent nationally. It is making an active effort to attract returning students who have obtained their Ph.D.'s or other advanced training abroad. The most prominent example is Chen Zhangliang, who was a top student at Washington University, where he studied with Roger Beachy; he could have obtained a permanent position in the United States. Peking University offered Chen several inducements to return, including an associate professor position, a large laboratory, and, perhaps most importantly, the chance to go abroad on an annual basis. Chen is currently supported by a 1.5 million yuan grant from the High Technology Program and a $30,000 grant from the Rockefeller Foundation. Chen's research focuses on the genetic engineering of resistance to viral diseases in plants, especially rice. Several other research projects at Peking University relate directly or indirectly to biotechnology. Zhang Longxiang's group focuses on comparative protein structure analyses and has determined the sequences of lactate dehydrogenase from the panda bear and C-reactive protein from a Chinese clam. Dr. Li's group is attempting to use somatic seeds to study somaclonal variation in plants and to propagate Chinese medicinal herbs. The protein structure groups of Gu Xiaocheng and colleagues are studying structure- function relationships of insulin, trypsin, and urokinase with an eye to eventual protein engineering. Although the department is one of the most dynamic in China, it also suffers from a high degree of inbreeding and from a top-heavy staff structure (features somewhat common in Chinese academic institutions). Whether the department's research accomplishments live up to its prestige and high funding priority or not will depend critically on the continuing recruitment of top-notch young scientists. CURRENT RESEARCH AT SELECTED INSTITUTES 52

SHANGHAI Fudan University Biotechnology research at Fudan University is predominately carried out at the Institute of Genetics, an academic department of the School of Life Sciences that performs research and graduate education but is not responsible for undergraduate students. The leader of the university's Institute of Genetics and of the School of Life Sciences is C.C. Tan, one of China's most highly regarded scientists and educators. Tan, who was a student of Thomas H. Morgan, received his Ph.D. from the California Institute of Technology in 1936 and returned to China in 1937. He was one of the discoverers of transposable elements in ladybugs; this discovery, together with a similar finding in corn, has allowed one of the basic frameworks for the development of genetic engineering to be formed. In China, Tan and his students continued their work on genetics, despite the complete disregard of this subject when Chinese life sciences were guided by Lysenkoism. Perhaps his greatest contribution was his role in influencing Chairman Mao to turn China from Lysenkoism. Agricultural production in China increased enormously because of his contribution in preserving classical genetics. Tan has received many awards and honorary degrees and is a foreign associate of the U.S. National Academy of Sciences. Research at the university's Institute of Genetics is carried out by 120 staff members and 93 graduate students. Total research support is 8 million yuan, making this, on a per capita basis, the most prosperous of all such research centers in China. These funds are derived primarily from 6.2 million yuan in High Technology Program grants, 960,000 yuan in Seventh 5-Year Plan grants, and 156,000 yuan in NSFC grants. The institute has also actively sought research support from abroad and receives approximately $100,000 from the Rockefeller Foundation and $70,000 from Interferon Sciences. In addition, the Fudan Foundation, an educational and lobbying organization based in Washington, D.C., has raised over $5 million and aims at raising another $10 million in order to establish an American studies center and the Thomas H. Morgan Science Center at Fudan University. It is hoped that the Morgan center, which is still in the initial planning stages, will provide an opportunity for Chinese scientists and students to cooperate with American scientists in a high- quality educational and research program. The section on microbial molecular genetics performs both basic and applied research on bacteria. The section chief, Sheng Zujia, received his Ph.D. from Columbia University and has a long and distinguished career in microbial genetics in China. Research support, mostly from the Seventh 5-Year Plan, amounts to 350,000 yuan. This section's basic research focuses on DNA replication in E. coli. Strains carrying a mutation in the dnaA gene, which codes for a replicase, are incapable of growing. This defe ct can be overcome by integration of a new CURRENT RESEARCH AT SELECTED INSTITUTES 53

replication origin from a plasmid or F-factor. Mao Yumin, an instructor who recently received his Ph.D. in this section, has made the surprising discovery that such integrative suppression is dependent on recA, a gene involved in DNA recombination. The effect of recA is highly dependent upon the integration site of the new origin in a manner that suggests that the recombinase allows DNA replication to proceed past a termination site. Control experiments show that this effect is dependent on the recombination activity of recA, not on SOS repair, and that it is independent of the type of replication origin. Current research focuses on proving the terminator bypass model by direct measurements of DNA synthesis and accumulation. The second major focus of the section on microbial genetics is the molecular biology of thermophilic bacteria, in particular, Bacillus stearothermophilus, which is capable of normal growth at 55°C. As might be expected, many of the enzymes from such thermophilic organisms are very stable at high temperatures, a useful property for industrial fermentation and biocatalysis processes. The gene for a thermostable α-amylase, potentially useful for direct fermentations from starch, has been cloned, partially characterized, and expressed in several vector-host systems. The cloning and preliminary characterization of heat-stable glucose phosphate isomerase, DNA polymerase, proteases, and lipases are also under way. At the same time, this group is carrying out more basic work on gene expression and genetic exchange in B. stearothermophilus in order to improve host-vector systems and facilitate genetic analyses. Promoter DNA sequences, which are required for high-level gene expression, have been cloned both from total DNA and from the glucophosphate isomerase gene. Vectors have been constructed using naturally occurring cryptic B. stearothermophilus plasmids and mutated, thermostable antibiotic resistance genes from E. coli . Two thermostable restriction enzymes, which may be important for genetic exchange, have been purified and characterized in terms of cleavage specificity. During the course of vector construction it was noted that a plasmid that expressed a foreign gene with high efficiency had picked up an extra piece of DNA from the chromosome. Subsequent studies showed that the extra DNA is one member of a family of transposons, a class of DNA element that is capable of excision and integration into the chromosome and extrachromosomal replicons. This transposon, which has now been marked with an antibiotic resistance gene, is already proving to be a highly useful genetic tool. Clearly, one goal of the B. stearothermophilus work, and the reason for its 300,000-yuan funding from the High Technology Program, is to produce thermostable enzymes for industry. But along the way there is the possibility of doing some very interesting basic work on protein structure-function relationships, the regulation of transcription, and mechanisms of genetic exchange in this unusual class of organism. The section of human and medical genetics consists of three groups under the leadership of Xue Jinglun, Zhao Shouyuan, and Chai Jianhua. Research support totals 2.7 million yuan, mostly from High Technology Program grants. The group CURRENT RESEARCH AT SELECTED INSTITUTES 54

of Xue Jinglun, who trained at Roswell Park Memorial Institute in Buffalo, New York, focuses on human gene therapy with the ultimate aim of curing inherited diseases by inserting a normal gene into the somatic cells of an affected individual. As a model system, they are studying hemophilia B, a blood-clotting disease caused by a mutation in the X-chromosome-linked gene for factor IX. They have transfected the factor IX gene, obtained from a group in England, into CHO cells and shown that it is expressed at the protein level. They have also, in collaboration with the Shanghai Second Medical University, established skin fibroblast cell lines from patients with hemophilia A and B. While good progress has been made, the ultimate success of this ambitious project will require many additional advances in delivery systems and understanding of factor IX gene regulation. The group of Zhao Shouyuan, who has been a visiting scientist at Yale University, concentrates on various problems relating to human cancer. Stomach cancer is much more prevalent in China than in other parts of the world. Zhao's group showed that stomach cancer cells express the Ha-ras oncogene and that DNA from these cells can neoplastically transform normal cells. However, they did not prove that Ha-ras is the active oncogene by DNA cloning; this was accomplished at another institute in Beijing. A second project is to characterize the regulation of and to overproduce lymphokines and cytokines, such as interleukin-2 and tumor necrosis factor, that may be useful in tumor therapy. Together with Li Changben, who trained for 2 years at the University of Maryland and 2 years at Yale, they cloned and sequenced the human interleukin-2 gene that had previously been described by Genentech. As a basic research topic, they are attempting to characterize the regulatory DNA sequences that control lymphocyte-specific expression of this gene by deletion analyses. In more applied work, they have successfully produced interleukin-2 in both bacteria and mammalian cells by genetic engineering. However, major improvements in yield and purification will be required to prepare sufficient amounts for human testing. The group of Chai Jianhua, who recently returned from 4 years at the European Molecular Biology Laboratory and Max Planck Institute in West Germany, is just starting an ambitious project to prepare a restriction and genetic map of the entire human X-chromosome. Their mapping will start at two known X-chromosome loci, those for hemophilia A and muscular dystrophy, for which probes have been obtained from other groups. Long-range restriction maps will be determined by an ingenuous ''jumping clone'' method whereby overlapping segments of DNA can be ordered without detailed characterization of the intervening sequences. The construction of the "jumping" and "linking" clone libraries from DNA of a multi-X-chromosome cell line is under way. The methodologies being developed in Chai's laboratory are potentially applicable to any chromosomal or organelle DNA. The institute's plant genetics section is divided into two groups working on the interrelated topics of cellular and molecular biology. The section is supported by 830,000 yuan from the Chinese government and $180,000 from the Rockefeller CURRENT RESEARCH AT SELECTED INSTITUTES 55

Foundation and Interferon Sciences. The cellular biology group, headed by Ge Koulin, focuses on methods for plant regeneration and gene transfer. They were among the first to succeed in protoplast regeneration of Phaseolus species, the common green bean that is an important food crop in China, and together with the sponsor, Interferon Sciences, have applied for a U.S. patent on this process. In common with many other groups in China and abroad, they have also regenerated rice from protoplasts of the Japonica but not the Indica strain. They are now in the process of DNA transformation of Phaseolus and other species with marker genes such as those for kanamycin resistance. Eventually, they hope to use this technique to improve food crops, but they have not yet decided the exact genes that they will use. The molecular biology group, under Wang Xunming, studies the structure and expression of plant chromosomal and organellar genes. In the past, they compared chloroplast tRNA genes of Vicia fabia and Brassica napus and showed that they have evolved at different rates. They also detected differences in the mitochondrial DNA of sterile male and maintainer rice strains, but the molecular basis of such variation was not determined. A current project is to obtain tissue-specific promoters to be used eventually for transgenic plant construction. The institute's genetic engineering section comprises three research groups led by Wang Qisong, Li Yuyang, and Zheng Zhaoxin. The general aim of the section, which is strongly supported by 3.3 million yuan in High Technology Program grants, is to produce useful proteins by recombinant DNA techniques. The group of Wang Qisong, who trained in Canada, focuses on methods for the synthesis and overexpression of genes encoding peptides. Using an automated DNA synthesizer, they have synthesized and assembled over 20 different genes including those for calcitonin gene-related peptide, which is potentially useful for control of hypertension; atrial natriuretic factor, which is also involved in blood pressure control; a foot-and-mouth virus coat protein peptide (a potential vaccine); and α-interferon and interleukin-2 (potential anticancer drugs). Most of these genes have now been expressed either in yeast or bacteria. A second project is to develop new methods for protein engineering through multiple, simultaneous point mutagenesis. This approach is being used to make novel hybrids between two drugs useful against myocardial infarction, namely, tissue plasminogen activator and urokinase. Finally, Wang has invented a clever new method for synthesizing mRNA from a synthetic DNA template. This RNA can be translated in vitro, thus providing a rapid assay method for genetically engineered enzymes and hormones. While many institutes in China now have DNA synthesizers, few, if any, are used as frequently and productively as they are in this laboratory. Li Yuyang's group is trying to use yeast to express various useful proteins such as HBsAg, atrial natriuretic factor, and calcitonin gene-related peptide. They employ strategies previously worked out in Western biotechnology companies, i.e., high-copy-number vectors, strong promoter sequences, and secretion signal leader sequences. A more novel strategy, which will be tested by a returning CURRENT RESEARCH AT SELECTED INSTITUTES 56

postdoctoral scholar, will be the use of the naturally high secreter strain Kluyveromyces lactis. Zheng Zhaoxin and associates work on genetic engineering in bacteria. They are particularly interested in the use of Corynebacterium glutamicum , an organism that is widely used for amino acid production and that possesses several advantages over E. coli for fermentation purposes, i.e., fast growth, safety, lack of proteases, and good secretion. They have constructed a Corynebacterium vector and established a transformation system; attempts to ferment a useful product are now in progress. This work is sufficiently novel to have attracted the attention of several European groups. A second project is to produce the calcitonin generelated peptide hormone which, in a collaboration with Peking Medical University, has been shown to reduce blood pressure within 90 minutes of administration. A major new effort revolves around Schistosoma japonicum, a liver parasite that is carried by rice paddy snails and affects three million people in China. Following Australian scientists' work on the Filipino parasite strain, two major antigen genes have been cloned and characterized. Overproduction of these proteins, and of corresponding monoclonal antibodies, should allow development of a reliable diagnosis method. Institute of Biochemistry (CAS) The Shanghai Institute of Biochemistry, established in 1958, is one of China's best known research centers. It was one of the only research institutes to remain active throughout the Cultural Revolution, during which important work on the synthesis of insulin and alanine tRNA was performed, and in the past 20 years, it has been at the forefront of bringing molecular biology to China. The recent history of the institute provides insight into the effects of China's new scientific policies on how research is conducted and supported at a major center. In 1984, CAS provided for all research costs, whereas now it is responsible for only about 10 percent. At that time, the institute was organized into several large divisions, each with about 40 people, and the pathway for funding was from institute director to division head to individual researcher. Now the institute has been reorganized into 37 much smaller groups, each responsible for obtaining its own funding. While the new system allows greater autonomy for individual scientists, it lacks the cohesiveness that made major projects such as the synthesis of insulin possible. Previously, the institute took great pride in its emphasis on basic research, but because of the new funding priorities, this stance has been abandoned. The decentralization and diversification of the institute's administration is evidenced by the opening, in 1988, of the Shanghai Molecular Biology Laboratory as a center for scientific training and international cooperation. Although this laboratory has its own director and publishes its own annual report, it is housed in CURRENT RESEARCH AT SELECTED INSTITUTES 57

the same building as the Shanghai Institute of Biochemistry and numbers among its members Lin Qishui, director of the institute. Plans are also afloat for two new national laboratories focusing on eukaryotic gene regulation and neuropeptides. One clear benefit of the new policies is increased attention to international cooperation. In 1987, the institute hosted over 300 foreign scientists and conducted numerous symposia, workshops, and minicourses (including two organized jointly by CSCPRC and CAS). The institute has a staff of 646, including 396 research and technical workers. It is located in the CAS Yueyang Road complex, in the old French quarter, adjacent to the Shanghai Institutes of Cell Biology, Physiology, and Materia Medica. Laboratories are located in a large, old, but well-equipped main building and a new wing devoted to molecular genetics. The group of Li Zaiping, who is well known in the West for his pioneering work on bringing molecular biology to China and promoting international cooperation, works on various basic and practical aspects of eukaryotic gene regulation and transcription. They have isolated and sequenced the 5.8S ribosomal RNA gene of the silkworm, Attacus ricini, and shown that the chromosomal copy contains a DNase-hypersensitive site under active transcription conditions. Interestingly, this site corresponds to a nuclease S1- sensitive site in the cloned, supercoiled DNA, perhaps indicating an underlying irregularity in the structure of the DNA. More recently, Li and colleagues have been studying the transcription of the HBV genome. They are using antisense RNA and antibodies to determine whether the "X-gene" is an autoregulator and are also searching for trans-acting factors encoded by the nuclear genome. A more applied project relating to HBV is the construction of vaccinia virus recombinants to be used as vaccines. This is similar to work in the United States and at the CAPM Institute of Virology. A vaccinia virus recombinant expressing HBsAg has been constructed and turned over to the Beijing Institute of Biological Products for scale-up. Recombinants expressing the pre-S1 and pre-S2 antigens, which contain binding sites for serum albumin, have also been constructed and will be compared with the shorter S-antigen for effectiveness. Yeast and E. coli have been used to express S- and X-fusion proteins as potential immunological screening reagents. In addition, fusion genes between the surface antigens of hepatitis B virus and hepatitis A virus are being constructed with the idea of making a multivalent hepatitis vaccine. Li's group is also pursuing several cancer-related projects. Several tumor growth factor fusion peptides have been expressed and shown, in agreement with results from the United States, to have increased antiviral activity. In collaboration with Japanese scientists, a study of antioncogenes has been initiated. Several different classes of those genes, each capable of suppressing transformation by a different set of oncogenes, have been cloned. The detailed characterization of these genes could yield important insights into the biochemical pathways of neoplastic transformation. CURRENT RESEARCH AT SELECTED INSTITUTES 58

One of the newer and most active staff members at the Shanghai Institute of Biochemistry is Hong Guafan, whose research focuses on the molecular biology of nitrogen fixation. Nodule formation is an essential prerequisite for symbiotic nitrogen fixation. In collaboration with workers at the John Innes Institute in England, Hong has identified a series of genes expressed by Rhizobium leguminosarum in peas. One of these, the nodD gene, was sequenced and expressed in E. coli, and it was shown that this protein specifically binds to the nod cluster intragenic region. While the initial phases of this work were done in England, students in Hong's lab in Shanghai are now actively pursuing the question of how certain organic compounds switch nodD from a repressor to an activator. In research initiated in Shanghai, Hong's group has also started to characterize the nod genes of fast-growing Rhizobium strains isolated from nearby fields. Following up on work started in Fred Sanger's laboratory at the Medical Research Council in Cambridge, England, Hong has also continued to improve DNA-sequencing methods by the use of a heat-stable DNA polymerase which can read through hairpin structures in the DNA at high temperatures. Several U.S. biotechnology reagent companies have expressed interest in this method. In order to entice Hong to return to Shanghai, CAS offered several incentives: a full professorship, an independent research group, a good-sized laboratory, research support, and the opportunity to go abroad annually to perform collaborative research. These were wise investments because Hong's group is certainly one of the strongest in China. Regular collaboration with Western scientists has been especially important, particularly during the early years when Hong was still setting up his laboratory and training students. Now workers in this laboratory have the opportunity, rare in China, to do original research on a current topic rather than simply repeat experiments done in the West. In a sense, the John Innes Institute provided the critical mass to establish an active research group in China. Structure-function relationships of insulin have been a major focus of the Shanghai Institute of Biochemistry since 1965. Using modified synthetic methods, several derivatives of insulin have been prepared and evaluated for biological activity and receptor binding. The results show that Phe26 plays a critical role in the structure of the hormone. The insulin A-chain has been expressed in E. coli, and several site-directed mutants are being worked up. In addition, various short peptide segments of insulin have been chemically synthesized and then tested for biological activities or the ability to compete for insulin inhibitors (i.e., inhibitor-binding sites). The conformation of these fragments has been probed by nuclear magnetic resonance methods, including nuclear Overhauser effect, with spinlattice relaxation time measurements. Many of the insulin experiments at the Shanghai Institute of Biochemistry overlap with those at the Beijing Institute of Biophysics; perhaps more rapid progress could be made by combining the Shanghai institute's expertise in synthesis and nuclear magnetic resonance methods with the Beijing institute's expertise in crystallography. CURRENT RESEARCH AT SELECTED INSTITUTES 59

Institute of Cell Biology (CAS) The Shanghai Institute of Cell Biology, founded in 1950 as the Institute of Experimental Biology, employs 370 personnel, including 50 senior researchers, 60 research associates, and 50 senior technicians. The institute carries out biotechnology research in several areas such as chromosome and chromatin biology, production of immunotoxins, and vaccine development using transgenic animals. This institute was one of the first to develop monoclonal antibody technology in China, and many of the basic and applied research projects use this methodology. The institute is also responsible for maintaining China's mammalian cell line bank. The chromosome biology group, in collaboration with colleagues at the Beijing Institute of Biophysics, is attempting to develop chromosome-like vectors for plants, especially rice. Using a novel antibody approach, they are trying to clone chromosomal DNA fragments containing centromere and telomere sequences. First, they prepared a panel of monoclonal antibodies directed against nuclear structures. Second, they determined the specificity of the antibodies by immunostaining nuclei; they also tested the function of these structures by inhibition studies. Lastly, in their current work, they try to pull out specific DNA fragments associated with these antigens. While the ultimate aim of the work is applied, it also involves a healthy dose of basic research on the structure and function of important chromosome components. A group under the leadership of L.C. Sze is attempting to use whole animals as bioreactors to produce valuable proteins such as HBsAg. In collaboration with Jiangsu Province Agricultural College, they injected a metallothionein-HBsAg hybrid gene into rabbit embryos and obtained 20 percent transgenic animals of which 10 percent produced detectable surface antigen in the blood. This work, supported by 1 million yuan from the High Technology Program and 1 million yuan from the Seventh 5-Year Plan, is being turned over to a biological products institute for further development. Consistent with the institute's traditional emphasis on immunology, several groups are using antibodies to detect and eventually fight cancer. An immunological kit for detecting α-fetoprotein, an early indicator for liver cancer, has been developed and marketed. Several monoclonal antibodies against hepatoma cell surface antigens have been isolated, and antibody-ricin immunotoxins have been produced in collaboration with the Shanghai Institute of Biochemistry. While these appear somewhat specific in vitro, their potential applications in vivo will require much further testing. Several members of the Shanghai Institute of Cell Biology attended the joint CSCPRC-CAS minicourse on immunotoxins in 1988, which, hopefully, will help them to design the appropriate experiments. Institute of Materia Medica (CAS) The Shanghai Institute of Materia Medica, founded in 1932, searches for new, physiologically active compounds and studies their structure-function relationships. CURRENT RESEARCH AT SELECTED INSTITUTES 60

The institute has over 400 staff, including 64 at senior levels. While members of the institute's staff are best known for their research on traditional Chinese medicines, they have recently begun to use the techniques of genetic engineering and molecular biology to produce and characterize new drugs. Antibiotic production and improvement is the main focus of a group led by Yang Shenti. They cloned, and overproduced in E. coli, penicillin G-acylase for use in the semisynthetic production of penicillin. The immobilized enzyme is now used commercially by several pharmaceutical companies in China. Attempts to improve the enzyme by genetic engineering are under way, but in the absence of basic information about the structure of the enzyme, it is unclear what residues to change. This group has also developed a microbial technique for producing threonine, a precursor for synthesis of the moxalactam series of antibiotics. In related research, this group is carrying out structural determinations on certain β-lactamase inhibitors which could potentially allow the use of penicillin and other β-lactams against normally resistant bacteria. A second project combining classical and modern approaches is a search for new neuropeptides from such sources as amphibian skin and gut, human brain, and certain tumor cells. Several new peptides have been isolated, sequenced, synthesized, and tested for biological activities, including analgesic effects. Attempts to produce some of these peptides by genetic engineering are just beginning. Several projects at the Shanghai Institute of Materia Medica concern the mechanism of action of anticancer drugs derived from Chinese medicinal herbs such as Camptotheca acuminata, a traditional Chinese treatment for leukemia. It was found that hydroxycamptothecin, the active ingredient, inhibits the expression of the ras and myc oncogenes while having little effect on fos or erb oncogenes. However, it is far from clear that the effect is either specific or the primary mode of action. The institute also produces the active components of several potential anticancer drugs by various methods including plant tissue culture; some of these are being tested in clinical trials in the United States in collaboration with the National Cancer Institute. Eventually, it is hoped that mutant cell lines that overproduce these drugs will be isolated. Institute of Plant Physiology (CAS) The Shanghai Institute of Plant Physiology is one of the best known institutes in CAS and has made many significant contributions in the field of plant physiology. Therefore, it was selected to house one of among a few plant molecular biology key laboratories. Designated as the Plant Biotechnology Laboratory, it was established in 1986 and occupies 2,500 square meters of space in the institute's new building. The Plant Biotechnology Laboratory was planned to have the capacity to house 60 scientists: 20 from Shanghai and the rest from elsewhere around the nation. Currently, there are several research groups, although the number can vary as the number and interests of visiting scientists fluctuate. The disease resistance and genetic engineering group, like other groups working CURRENT RESEARCH AT SELECTED INSTITUTES 61

on genetic engineering for plant disease resistance, focuses on plant viruses. However, this group is attempting to use a different coat protein gene from TMV and a different experimental strategy than the other groups. The results are not yet available for evaluation. However, they anticipate that deletion of the bases coding for polymerase can suppress the synthesis of the TMV coat protein subgenomic RNA. If this is successful, TMV infection can be prevented instead of delayed. In addition, this group also initiated a project to attack diseases caused by fungi. They plan to isolate the components from a rice cultivar sensitive to Pyricularia oryzae. Currently, they have isolated a protein from Gastrodia elata that may contribute to the resistance of such fungal disease. The leader of this group is C. Wang, who joined the institute very recently. He has received substantial financial support, but the lack of trained and experienced workers is proving to be a major obstacle. The primary goal of the regulation of gene expression group, led by the well-known professor, M.M. Hong, is to study the organization, expression, and regulation of the waxy gene from rice. In rice (Oryza sativa), the waxy gene is located on the number one chromosome. The starch granule-bound uridine diphosphate-glucose starch transferase is the catalyst for the synthesis of α- amylase in the pollen grain and endosperm. Since α-amylase is synthesized only in these tissues, the expression of the waxy gene is tissue and developmental stage specific. Therefore, this group is working on the cloning, isolation, and sequence of this gene. It is intended to study the coding as well as the regulatory regions of this gene. It is hoped that the cis-acting elements as well as the trans- acting factors can be identified. This is a very important step in the attempt to improve the quality of rice. At the same time, this group is also interested in studying the cause of differences in α-amylase content between Japonica and Indica strains of rice. This may be related to the regulation of expression of the waxy gene. Furthermore, the lack of α-amylase in some cultivars of rice may reflect the lack of expression of the waxy gene. This group has constructed four rice genomic libraries since 1987. Using the waxy gene probe provided by Nina Fedoroff at Johns Hopkins University, they have proceeded expeditiously in identifying the waxy genes. The cell and tissue culture group reflects the institute's traditional strength in this area. Currently, there are four areas of investigation under the leadership of C.H. Xu, the deputy director of the institute: 1. In vitro clonal propagation: Using in vitro techniques, Xu's group has regenerated over 70 plant species. Many of them are important crop species including rice, wheat, maize, sorghum, soybean, cabbage, and rape. It should be noted that the orchid derived from the in vitro clonal method flowers in a few months compared with 5 years for orchids derived from seedlings. 2. Protoplast fusion and cell hybridization: They have obtained over 10 plant CURRENT RESEARCH AT SELECTED INSTITUTES 62

species derived from fused protoplasts. Among them are some important crop species such as rice, soybean, and tobacco. 3. Mutation induction: They have focused on the selection of mutants with a high concentration of essential amino acids or with new stress resistances. Tobacco plants which can tolerate 2 percent sodium chloride and soybean cells resistant to glyphosate (the active ingredient in Roundup, an herbicide made by Monsanto) have been selected and regenerated into plants. 4. Cell transformation: Using Ti plasmid and reporter genes, they have obtained a series of transformed plants, but no novel contributions are forthcoming. The cotton disease resistance group, which is led by G.Y. Zhou of the Shanghai Institute of Biochemistry, will be administratively, if not physically, transferred to be part of this open laboratory. For many years, Zhou's group has worked on a technique for the introduction of foreign DNA from disease- resistant cotton species into disease-sensitive cotton species. They take advantage of the pollen tube pathway and inject foreign DNA into the embryonic sac to transform the zygote. This approach has encountered technological difficulties as well as collegial criticisms. In collaboration with the Jiangsu Province Institute of Industrial Crops, they have obtained some cotton hybrids expressing disease-resistant traits. In addition, there is a group working on B. thuringiensis toxin and a group working on storage proteins. GUANGZHOU Guangdong Agricultural Academy of Sciences In 1987, the Center for Agricultural Biotechnology was established in the Guangdong Agricultural Academy of Sciences. It is mainly an organization that coordinates existing activities loosely related to the broad definition of biotechnology. Currently, there are 54 staff members, 34 of whom are scientists at different levels; 1 senior, 6 associate, and 13 assistant staff scientists; and 14 technicians. According to their plan, the center will have four divisions: gene, cell, enzyme, and fermentation biotechnology. To do this, additional scientists have to be recruited, which will not be easy. Most research activities at the present time are centered around tissue culture work for the purpose of propagation, e.g., banana and pineapple. Surprisingly, great effort has been directed to the breeding of seedless watermelon, which was successfully achieved almost 30 years ago in Taiwan. Obviously, this center is in its infancy, and it will be a long time before it can move into molecular aspects of agricultural research. However, this center is an excellent resource for improving rice crops: they are studying a unique strain of CURRENT RESEARCH AT SELECTED INSTITUTES 63

black rice that is claimed to be more nutritious than the ordinary strain and to possess other values. South China Agricultural University South China Agricultural University attained its university status in 1984. Currently, there are 10 academic departments including most of the classical agricultural disciplines such as agronomy, horticulture, animal husbandry, veterinary medicine, agricultural engineering, and forestry. Among the 820 faculty members, there are 240 professors and associate professors, 480 instructors, and 100 teaching assistants. The undergraduate population is 3,000. The university also offers graduate degrees at both M.S. and Ph.D. levels in highly selected areas. In addition, there are 36 foreign students, mostly from other developing countries. The current administration is interested in promoting and strengthening research activities within the university. Under the present organization, there are 20 research units, one of which is genetic engineering. The others include crop genetics and postharvest physiology. The university has a very large collection of germplasm, including over 7,000 cultivars of rice. Currently, there are six faculty members in the research unit of genetic engineering: two associate professors, one senior research staff, and three research associates who focus on two areas. In animal sciences, the emphasis is on growth hormone and antibacterial peptides. In the plant sciences, rice and rape diseases are the major interest. This unit is currently supported by grants from the Seventh 5-Year Plan, NSFC, and donations from various sources in Hong Kong (HK$500,000; HK$7.8 = US$1). South China Institute of Botany (CAS) The South China Institute of Botany, established in 1929, was originally known as the Institute of Agriculture and Forestry and was affiliated with Sun Yatsen University. It was reorganized in 1954 and transferred to CAS, where it was expanded from one taxonomy department to include other botanical disciplines. Currently, it has six departments and a botanic garden and is staffed with 568 workers at different levels. Among them are 334 scientists or technicians, 10 professors, 65 associate professors, 3 senior scientists, 10 senior engineers, and 25 M.S. graduate students. The six departments and their respective focuses are listed below: 1. Taxonomy: classification and phylogenetic studies of many plant families aided by chemotaxonomy, cytology, anatomy, and polynology are actively carried out. 2. Phytomorphology: includes anatomical studies on weed structure, embryology, and pollen morphology. CURRENT RESEARCH AT SELECTED INSTITUTES 64

3. Phytochemistry: surveys the chemical compounds extracted from a large number of plants for useful or valuable substances such as the effective antitumor compounds Harringtonine and Homo-Harringtonine. 4. Ecology: major studies include ecosystem, pollution ecology, and the use of artificial plant association for the regeneration of Chinese plant species. 5. Physiology: investigates stress adaptation, seed and postharvest physiology, and tissue culture and protoplast fusion. 6. Genetics: the primary emphasis of this department is on biotechnology to study heterasis and cytoplasmic male sterility. This department invested heavily in rice breeding for high-quality, high-yield, and disease resistant crops. The institute's biotechnology program is carried out at the interdepartmental level. The scientists involved are primarily from three units: genetics, physiology, and botanic garden. This biotechnology group is moderately supported by grants from the High Technology Program, Seventh 5- Year Plan, and NSFC at about 200,000 yuan per year, plus $10,000 from the Rockefeller Foundation to support research on rice. Like so many other groups in China, they started in the early 1960s with cell and tissue culture methods. In fact, most activities related to biotechnology are centered around the production of plantlets by tissue cultivars for commercial purposes. The micropropagation group is large and consists of over 40 people, including two professors and six associate professors. In the protoplast fusion groups, they have produced eggplant and potato hybrids that had already been achieved in the West. Obviously, there is need for more molecular biologists in this program. One scientist, Huang Yuwen, just returned from abroad. She is in the process of setting up a molecular biology laboratory to work on rice cytoplasmic male sterility. But progress is extremely slow, a frustration faced by almost everyone who returns from overseas. Zhongshan (Sun Yatsen) University Zhongshan University was established in 1952 by combining certain academic departments in basic and social sciences from two well-established universities (Ling Nan and Sun Yatsen). Currently, it has over 30 academic departments and institutes, including basic science departments in biology, chemistry, physics, mathematics, computer sciences, electronics, mechanics, geography, and geology. The student population has just reached the 9,000 mark. In addition, there are over 100 graduate students, 10 percent of whom are Ph.D. students. The faculty-to-student ratio at Zhongshan University is roughly 1:7; there are about 120 professors, 350 associate professors, 700 instructors, and 200 teaching assistants. In 1986, the Center for Biotechnology was established at Zhongshan University by organizing into a separate administrative unit existing faculty members whose research interests were related to biotechnology. It was initially funded by 1.2 million yuan from SEDC and 200,000 yuan from Guangdong Province. The CURRENT RESEARCH AT SELECTED INSTITUTES 65

director of the center is Li Baojian, a plant molecular biologist who has recently returned from Cornell University. The plant genetic engineering group is the largest in the center. There are two professors, seven associate professors, five instructors, and 24 research associates at the M.S. level being supported by 120,000 yuan from the High Technology Program and the Seventh 5-Year Plan. Since this group has been actively working on rice, a $30,000 grant from the Rockefeller Foundation has proved to be extremely helpful. The research subjects of this group are rather diffuse and include gene transformation, cell fusion, artificial seed, and micropropagation. Formation of a highly focused program should be the top priority. One area of considerable interest is the attempt to locate and to clone the gene that is believed to play an important role in the wide compatibility of rice cultivars. It was good news to learn that they were successful in developing artificial seeds, but disappointing to learn that they failed to keep them. Another notable group in the center is the microbial genetic engineering section. It is staffed by two full professors, three instructors, three research associates, and seven graduate students. This group's support is primarily provided by the Seventh 5-Year Plan, NSFC, and Guangdong Province at 120,000 yuan annually. Its main focus is on the construction of vectors from Bacillus species, cloning of industrially useful genes, and the isolation of heavy metal resistance plasmids. One unique project, which is just under way, deserves special mention. They have isolated a plasmid from bacteria found in the gut of an insect that grows on rice. This plasmid may have the potential to be used as a biological control agent because it kills some pathogens when it is transferred into E. coli. This center is a very well-coordinated, multidisciplinary organization that includes biologists, chemists, physicists, and engineers. The petrochemical engineering group is very active, and they use immobilized enzymes for scaled up production. This is a new and energetic group that has the potential to advance rapidly. TIANJIN Nankai University Nankai University, located in the major port city of Tianjin, is one of China's national key universities. Biotechnology research is carried out in the Department of Biology and the Institute of Molecular Biology. The Department of Biology, founded in 1971, is staffed by 170 people, including nine full professors and 42 associate professors. There are 450 undergraduate students, 66 M.S. candidates and four Ph.D. students. Total research funding is approximately 550,000 yuan per year, mostly from Seventh 5-Year Plan grants. SEDC pays staff salaries and 20,000 yuan for each Ph.D. student's research costs but provides no direct research grants. The Department of Biology is divided into seven groups focusing CURRENT RESEARCH AT SELECTED INSTITUTES 66

on molecular biology, biochemistry, microbiology, genetics, enzymology, plant physiology, and marine biology. The department publishes approximately 30 articles a year, all in Chinese. The university's Institute of Molecular Biology was founded in 1983 to provide a center for advanced biotechnology research at Nankai. There are 50 staff members, including nine full professors, together with 75 M.S. students and 21 Ph.D. students. The institute is supported by 400,000 yuan per year from three High Technology Program grants, nine Seventh 5-Year Plan grants, and several awards from industry and from Tianjin City. Fifteen of the institute's Ph.D. graduates have been sent abroad for postdoctoral training, and all of them apparently have returned. The institute is housed in its own well-equipped 4,500-squaremeter building. Much of the biotechnology research focuses on microbiology with potential industrial and agricultural applications. A gene for heat-stable α- amylase has been cloned from a thermophilic bacterium. A naphthalene- degrading plasmid from Pseudomonas aeruginosa has been transferred to E. coli . Toxins from Bacillus sphericus (specific for mosquitoes) and B. thuringiensis (specific for cabbage worms) have been characterized, and attempts at gene cloning and transfer are under way. While lacking the vitality of Beijing or Fudan Universities, it is still regarded as one of China's best educational institutions and attracts top-notch students. (It has also built a strong reputation in mathematical sciences, largely through the efforts of the famous Chinese-American mathematician, S.S. Chern.) The fact that biological research has gone from essentially no support to nearly 1 million yuan per year, all within 5 years, speaks to the influence of the new granting policies throughout China's educational system. However, judging from the several projects reviewed during a brief visit, it does not appear that competition for this funding has lent any special urgency or inventiveness to the research process itself. CURRENT RESEARCH AT SELECTED INSTITUTES 67

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