Present status of genetic engineering and biotechnology in South Korea.

related to biotechnology such as molecular biology, genetics, microbiology, biochemistry, and genetic engineering in the last year or two, partially because of demand for biotechnologists from the industries and partially because of the perspectives to biotechnology in the near future. Traditionally, university-industry research cooperation has had some barriers because of benefits to industry and university balance sheets but recently the cooperation in the field of biotechnology has been increasing in the last few years because of demand of basic knowledge from the industries. The number of publications related to new biotechnologies has been increased dramatically from 17 in 1983 to 172 articles in 1988 (Table 2).

Present Status of Genetic Engineering and Biotechnology in South Korea Moo Je Cho

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Otago on 12/29/14 For personal use only.

1. INTRODUCTION Biotechnology, applied biological science, is a rapidly growing interdisciplinary technological field based on the principles of life sciences such as microbiology, biochemistry, and molecular biology. In Korea, there is increasing recognition of the future potential of biotechnology in academic institutions as well as in industries all around the world due to several underlying reasons: (1) the need to develop and conserve our national resources of materials and energy, (2) the need to control increased pollution and wastes for clean environment and (3) the opportunities afforded by advances in science and technology to make heretofore impractical biochemical processes economically feasible. Research and development in this field have rapidly progressed in Korea during the last few years especially in the field of application of new biotechnology techniques such as recombinant DNA techniques, hybridomas, embryo transfer, cell culture, and cell fusion. The number of scientists majoring in molecular biology, microbiology, and biochemistry, and research funds supported by government and private companies are increasing dramatically (Table l).'-3

Table 2 Number of Publications Related to New Biotechnological Research Published in Korean Journals Categories Gene splicing Hybridomas Cell culture and fusion Protein engineering Total

Table 1 Trend of Fund Increase Supported to New Biotechnology Research (Unit: $1,000) Fund source

1983 1984

1985

1986

1987

1988

Increasing' index (%)

Government Company Total a

2,860 4,143 5,636 6,143 8,143 10,143 6,857 16,571 22,429 22,714 27,430 33,871 9,717 20,714 28,065 28,857 35,573 44,014

355 494 453

Increasing index compared to 1983.

A growing array of companies, including giants like Lucky, Samsung, and Daewoo, have established research institutes for biotechnology, and 19 of them have assembled the Genetic Engineering Research Association committing themselves for the further development of genetic engineering and biotechnology.4They are mainly interested in the production of pharmaceuticals for the human health care but some of them are even interested in plant biotechnology like production of secondary metabolites by plant cell culture and artificial seed production.5.6 More than 15 universities have established new departments

1983 1984 1985 1986 1987 1988

17

17

23 2 6

39 2 14

76 11 24

31

55

111

91 12 19 1 128

125 18

26 1 172

In view of the importance of research and development endeavors in biotechnology, Korea has launched the national program of genetic engineering and biotechnology through all possible means and ways to solve national problems for the development of food resources and alternative energy as well as means of environmental pollution and disease control. The Korean government has established Genetic Engineering Center affiliated KAIST, while the National Assembly has adopted a bill for the promotion of research and development in genetic engineering to establish a fm institutional structure. Although research and development endeavors of Korea in biotechnology have started late and are smaller in scale than the other developed countries, the Korean government and industries as well as scientists and technologists in allied technological disciplines are enthusiastic in building up research capability, technological know-how, and the productivity of bio-industries in Korea. In this paper, recent progress in the research and development related to new biotechnology techniques in Korea is reviewed.

Moo Je Cho earned a B.S.at Gyeongsang National University, ChinjuSi, South Korea; an M.S. at Seoul National University, Seoul, South Korea; and a Ph.D. at the University of Missouri, Columbia. Dr. Cho is Director and Professor, Genetic Engineering Institute, Gyeongsang National University. Chiniu-Si. South Korea.

1990

47

Critical Reviews In II. BIOTECHNOLOGY INDUSTRIES IN KOREA


Fermentation industries in Korea have been continuously developed during the last quarter of a century. At the early stage of the industrial development, several fermentation industries were only capable of producing fermented foods such as soy sauce and rice wine, which became the basis of producing amino acids, enzymes, and antibiotics. The total production value of fermentation products in Korea was about 2 billion dollars (1980) which was equivalent to 3.8% of the total GNP in the same year (Table 3).4

Table 3 Products of Bio-Industries in Korea (1980) Products Alcoholic beverage Fermented soy bean products Kimchi Dairy products Amino acids Antibiotics Mushrooms Starch sugars Enzymes Biological products Vitamins and organic acids Total (A) Total GNP (8) AiB x 1 0 0 ( % )

Items Antibiotics Vaccines Hormones Imitated bloods Diagnostic reagents Total

1981

1982

1983

203,271 257,820 312,587 21,368 26,496 42,884 24,179 31,084 33,556 13,923 16,989 20,789 900

1,651

1984

1985

345,337 351,154 102,368 90,899 38,389 41,074 25,381 26,690

1,991

2,949

Increasing" index (Yo) 72.8 325.4 69.9 91.7

3,506

289.3

263,641 334,040 411,807 514,424 513,323

94.0

' Increasing index compared to 1981.

Amounts ($l,OOO) 1,286,000 32,800 34,300 168,500 77,100 165,700 57,100 5 1,400 14,300 18,600 700 1,906,500 5 1,520,000 3.81

The major fermentation products in Korea are alcoholic beverages and fermented foods. However, the production of amino acids such as monosodium glutamate (MSG) and lysine can be recognized as major fermentation products produced by use of the modem microbial technology. Pharmaceutical production also plays an important role in the development of Korean bio-industries. The total production value of pharmaceutical products in Korea has gradually increased from 260 million dollars in 1981 to 5 10 million dollars in 1985, 70% of which is contributed by biomedical products such as antibiotics, blood fractions, and vaccines (Table 4).' Antibiotics are the most important therapeutic drugs produced in Korea and the production technology has been developed step-by-step by domestic pharmaceutical companies since 1970. At present, five major antibiotics such as tetracycline, kanamycine, rifamycin, erythromycin, and gentamycin have been locally industrialized (Table 5). As mentioned earlier, recently nineteen companies including leading pharmaceutical, food, chemical, and brewery companies installed the Korea Genetic Engineering Research Association in 1982 to make a cooperative effort for the development of genetic engineering and biotechnology in Korea. The special interests of the participants are summarized in Table 6. 48

Table 4 Production of Biomedical Product in Korea Related to Biotechnology (Unit: $1,000)

Table 5 Fermentation Products and lndustrles in Korea (1980) Products Alcohol

Organic acids MSG Lysine Phenylalanine Nucleic acids (AMP, GMP) Enzymes a-amylase Glucoamylase Protease Lipase Antibiotics Tetracycline Kanamycine Rifamycin B Erythromycin Gentamycine Polysaccharides Xanthan gum

Production companies Jinrho, Pughan Fermentation, Donglip Industries, Bohae Industries, Pungkuk, Yuwon Industries Seoul Miwon Co., Ltd., Cheil Sugar and Co. Seoul Miwon Co., Ltd. Cheil Sugar and Co., Ltd. Seoul Miwon Co., Ltd., Cheil Sugar and Co., Ltd.

Pacific Chemical Industrial Co., Ltd. Dong-A Pharmaceutical Co., Ltd. Dong-A Pharmaceutical Co., Ltd. Pacific Chemical Industrial Co. Pacific Chemical Industrial Co. Chong Kun Dang Corp. Dongmyong Industries Yuhan Chemicals, Chong Kun Dang Corp. Chong Kun Dang Corp. Chong Kun Dang Corp. Shinhan Milling Co.

Korea Steel Chemical Co., Ltd., a venture laboratory founded by Daewoo, is producing 30 kinds of restriction endonucleases and other DNA manipulating enzymes. Korea Green Cross Co. and Cheil Sugar and Co., Ltd. are producing Korean type hepatitis B virus (HBV) vaccine by using new biotechnologies and phenylalanine produced by the recombinant DNA technology to be commercialized by M i ~ o n . ~ , ~ The major technology for the commercialization of bio-products has been developed by KAIST and several universities. However, the research and development capability of the industrial sector in Korea has been gradually built up during the last several years so that considerable technological breakthrough and industrialization of bio-products could take place

Volume 10, Issue 1

Biotechnology Table 6 Biotechnology Research Institute or Laboratory Founded by Companies and Their Special Interests Name Korea Green Cross Corp. Oriental Brewery Co., Ltd.


Korea Steel Chemical Co., Ltd. KOHAP Ltd. Korea Chemical Co.. Ltd. Dong Shin Pharmaceutical Co., Ltd. Dong-A pharmaceutical Co., Ltd. Lucky, Ltd. Seoul Miwon Co., Ltd. Yukong Limited Yuhan Corporation

I1 Dong Pharmaceutical Co., Ltd. Cheil Sugar and Co., Ltd. Chong Kun Dang Corp. Kolon Industries, Inc. Pacific Chemical Industrial Co., Ltd. Hankuk Yakult Milk Products Co., Ltd. Korea Explosives Co., Ltd. Young Jin Pharmaceutical Co., Ltd.

Table 7 Major Technological Development in Biotechnology Year

Special interests

1974 1974

HBV-vaccine, human monoclonal antibodies, test kits (HB,AIDS, etc.) Yeast breeding, embryo transfer, virus free hop Restriction endonucleases, hyaluronic acid, cephalosporin Biopilotplant system Herbicides, plant tissue culture TNF (tumor necrosis factor) HBV-vaccine, glutathione, test kits (HV, AIDS) Interferons, growth hormone, colony stimulating factor Phenylalanine, lysine, aspartame Biopolymer, lysine, aspartame Proteases, collagen, human chorionic gonadotropin Anticancer agent, Bacillus fhuringensis toxin, lactic acid Interferons, TPA, HBV-vaccine Rifamycin, erythropoietin, cyclodextrin Human gastric cancer test kit, steroid, biopolymer Collagen, cyclodextrin, bioactive substances from plant Anticancer agent, protoplast fusion, lactic acid Cephalosporin, Bacillus fhuringensis toxin, antibiotics Pharmaceuticals

1975 1975 1976 1977 1977 1978 1978 1979 1979 1980 1980 1980 1980 1981 1982 1983

in the industrial sectors with their own research and development capabilities (Table 7).

1986

111. GENE SPLICING

1986

Development of the recombinant DNA techniques represents the major breakthrough not only in molecular biology but also in applied biology called biotechnology. A major objective of the techniques is to insert a foreign gene for a desired product into an organism under conditions such that the foreign gene will be expressed more abundantly than the native genes. A useful gene can be cloned from all the forms of organisms, both prokaryotes and eukaryotes, or can be synthesized chemically according to the information obtained from the peptide sequences. Since Lee et cloned Pstl restriction endonuclease gene into pBR322 plasmid vector from Providencia stuartii 164 in 1982 and Kim et al.9 separated tRNA genes from Aspergillus nidulans in 1983, the research using the recombinant DNA techniques has been dramatically developed in Korea. Many genes coding for compounds with medical or industrial

1986 1986 1987

Technology or product developed Production of tetracycline Production process of SCP from hydrocarbon Production process for high fructose corn syrup Production process for high fructose corn syrup Purification process of urokinase Fermentation process of GMP and IMP Enzymatic hydrolysis of RNA Fermentation process of xanthan gum Glucose test strip

Industries for commercialization

Research institute

Chong Kun Dang Corp. KAIST

Lucky, Ltd.

KAIST

Seoul Miwon Co., Ltd. Korea Green Cross Corp. Cheil Sugar and Co., Ltd. Seoul Miwon Co., Ltd. Shinhan Milling Co.

KAIST

Yeong Dong Phanna- KAIST ceutical Corp. Artificial diet for silkworm Dong Bang Oil Co. KAIST Fermented feeds using agri- Used by farmers KAIST cultural wastes KAIST Fermentation process for ri- Yunhan Chemicals famycin B production Fermentation process for ri- Chong Kun Dang Corp. KAIST famycin B production Erythromycin Chong Kun Dang Corp. Gentamycin Chong Kun Dang Corp. Pregnancy test kit Yeong Dong Pharma- KAIST ceutical Corp. a-Interferon Korea Green Cross Corp. Korea Green Cross Hepatitis B vaccine Corp., Cheil Sugar and Co., Ltd. Aspartame (production of Cheil Sugar and Co., phenylalanine) Ltd., Korea Green Cross C o p . Diagnostic reagent-HBV Cheil Sugar and Co., hepatomer Ltd., Korea Green Cross C o p . Microbial pesticide and soy- Korea Explosives Co., KAIST bean inoculant Ltd. Prothrombin (blood clotting Lee Yun Chemicals KAIST Corp. agent) Interleukin-2 (anticancer) Clinical test KAIST

interest have been cloned from microorganisms, plants, and animals including human as indicated in Table 8. Some of them are sequenced (Table 9) and gene organization and regulation mechanisms of the gene expression are characterized. Several vectors for the introduction and expression of the foreign genes into specific hosts were constructed (Table 10). The research using the recombinant DNA techniques is mainly focused on the production of medical products like vaccines, growth hormones, tumor necrosis factors (TNF) including interferons and interleukin, antibiotics, and diagnostic reagents.

1990

49

Critical Reviews In Table 8 The Cloned Genes Kingdom Microorganism

Genes cloned

Source of gene N . crasa

10

a-amylase Thermostable a-amylase

B. amyloliquefaciens B. srearoihermophilus B. licheniformis ATCC278 1 1 Alkalophilic Bacillus spp. Penicillium varuculosum Alkalophilic Bacillus spp. B. subtilis Cellulomonas biaiorea Alkalophilic Bacillus spp. K17 Zymomonas mobilis Srrepiomyces phaeochomogenes E. coli K . pneumoniae NFB-320 Bacillus spp.

11.12 13,14 15

P-glucosidase Glucose isomerase Xylose isomerase Pullulanase Raw starch digesting enzyme Alkaline protease Alkalophilic protease 3-isopropyl malate dehydrogenase Glutamine synthetase Homoserine dehydrogenase Hydrogenase Pectate lyase RAD4 RNA 1 Ski2 HIS, ADE 1 Aspartokinase aah (DNA repair) sep (cell division) trpL(Aff) frpEbBR Acetyl COA carboxylase

16 17 I8 19 20 21.22

Plant

23 24 25,26 27,28 29 30

S . cerevisiae S. cerevisiae

34 35

31 32 33

36,37 38 39

40 41 42 43 44 45,46 47 48,49 50 51 3 2 53 54 55.56 57.58 59,60 61 62 63 64-47 68 69 70 71

Sraphylococcus aureus DH- 72 1 Bovine 13 Human 74 Bovine 75 Rat liver 16

Human

71

Human Human Human Drosophila Human (synthesized) Human (synthesized) Human (synthesized) soybean Pea Potato Alfalfa

78.79 80 81 82 120 120 292 83 84 85 86

Table 9 DNA Sequenced Genes Genes

Strepromyces spp. Corynebacrerium spp. Bacillus spp. Kluyveromyces fragilis

E . coli Bacillus spp.YA- 14 S . cerevisiae S. cerevisiae S . cerevisiae S . cerevisiae S. cerevisiae S. cerevisiae E. coli E. coli E . coli LCllO E . coli tR"4ASP A . nidulans tRNA,, A. nidulans 17s rRNA Teirahymena ihermophila hmr3 HBsAg(Pre-S,) Hepatitis B virus HBsAg(hepatitis B virus Hepatitis B virus surface antigen) Reverse transcriptase Avian Sarcoma virus nif gene cluster K . pneumoniae Enierobacier agglomerans nif genes a-acylamino-p-lactam Acerobacrer rubidans acylhydrolase Crystal toxin protein Bacillus rhuringenesis var. kursraki HD-1 Subtilisn B. licheniformis bdi methylase Breuibacrerium divaticarum FERM5948 E. coli ATCC 1 I105 Penicilline G acylase Streprococcus equisimilis Streptokinase

50

Ref.

a-amylase

Alkaline amylase Cellulase CMCase P-glucanase Cellobiase P-xylosidase


Animal

Chloramphenicol resistance Growth hormone Growth hormone Pepsinogen(cDNA) Lactate dehydrogenase A Epinephrine synthesizing enzyme Interferon-y (cDNA) Interleukin-2 (cDNA) Osteocalcin Suppressor[Su(S)*] Insulin A chain Insulin B chain Roinsulin Glycinin Chitinase Proteinase inhibitor-I1 Leghemoglobin

ARS (partial) Cellulase Hydrogenase MAK 18 P-xylosidase HBsAg HIS, Proteinase inhibitor4 tRNA"' tRNA"P 5srRNA hsp7O

Source Candida tropicalis B. subrilis E . coli S . cerevisiae Alkalophilic Bacillus sp. K-17 Hepatitis B virus S. cerevisiae Potato Human A . nidulans Pseudomonas purida Chaeiomorpha moniligera Drosophila melanogasrer

Ref. 87 88 37 89 22 90-Y 1 92 85

93 94

95 96 97

The surface antigen gene of Korean type-hepatitis B virus (subtype adr) was extensively studied in order to produce HBV vaccine by the recombinant DNA m e t h ~ d . ~ ~ - ' ~ .~',' ''-'*l4~ because HBV causes serious human liver disease and the disease is one of the major infectious viral diseases in Asia and Africa. The structural gene of HBV surface antigen (HBsAg) was cloned into expression vector for yeast, which consists of the promoter of galactokinase gene and the signal peptide gene of killer toxin as a secretion signal. Cheil Sugar Co., Ltd. is selling HBV-vaccine produced by the cloned HBsAg gene in yeast, and the vaccine named Hepaxin B was selected as a new chemical entity (NCES) of 1987 in the journal Scrip published in England. Another vector which can be replicated in higher animals, pKSVML-TKp, was constructed with replication origin of SV40 and ~ B R 3 2 2 . HBsAg l~ gene was cloned in the vector and introduced into thymidine kinase-deficient mouse L-cell, where the gene expressed mouse L-cell could be selected. lo9 A cDNA for human interleukin-2 (IL-2 or T-cell growth factor), a lymphokine synthesized and secreted by some T-

Volume 10, Issue 1

uiotecnnoiogy Table 10 Vectors Constructed for Special Purpose Name

Function

pBR322, Trpl (yeast trypto- Yeast-E. coli shuttle phan marker gene), OR vector (yeast 2 pm origin of replication), CEN3 (centromere from yeast chromosome 3), ARSl (autonomous replicating sequence from yeast chromosome 4) pYER pBR322, Trpl, 2 pm OR, Yeast-E. coli shuttle vector ARS 1 pBR322, Trpl, 2 pm OR, Yeast-E. coli shuttlepYEC CEN3 vector pBBR322, origin of replica- Zymomomas-E. coli pHZ22 tion from pZM3 (3.9 kb in- shuttle vector digenous plasmid from Zymomonas mobilis ATCC 10988) Zymomonas-E. coli RP4, pHZ22 pHZ224 shuttle vector pBR322, origin of replica- Zymomomas-E. coli pBZ4 1 tion from pZM886 (1.7 kb shuttle vector indigenous plasmid from Z. mobilis ATCC10988) pRL81,82 pBR322, N gene and PL pro- Expression vector in E. coli 83,84 moter of X-phage pHW25,27 pBR322, pSBK203 (3.7 kb E . coli-B. subrilis shutindigenous plasmid from tle vector 33 Staphylococcus aureus) pBR322, pASR286 (57 kb Rhizobium-E. coli shutpGUR15 tle vector indigenous plasmid from Acacia rhizobia sp86) pBR322, XP,promoter, cIts Conditional expression pPLl11 repressor from cI857 vector in E. coli Vector in Candida pUC18, ARSl, Trpl puc-ARS rropicalis Trp Blunt end DNA cloning pBR322 derivative pRSRl16 vector pBR322, pUBl10 pC7 (3.0 Corynebacrerium-E. coli pJEC7 Kb indigenous plasmid shuttle vector from Corynebacrerium glufamicum ATCC13058) pCT30,20 tac promoter coding seOverproduction vector quence of N-terminal porin E . coli tion of P-galactosidase, polylinker of region of 3'end lacZ pSVML-TKp pBR322, origin of replica- Expression vector in tion and promoter of SV40, mammalian cell thymidine kinase gene from herpes simplex virus pRE18 pUCl8, Yeast-E. coli shuttle vector pRN18 ARSl from S. cerevisiae Vector in S. cerevisiae pIKS YIp5, A R S from Cundida fropiculis IF005 18 pYECR


Characteristics

Ref. 98

98 98 99

99 100

101 102

103

104

105 106

107

108

109

110 111

cells following activation with antigens or mitogens, which has attracted a great interest for clinical potential in cancer immunotherapy was cloned in expression vector containing XpL

promotor. A high level expression of IL-2 was obtained in E. coli harboring IL-2 cDNA under the control of the XpL promoter. The recombinant human IL-2 (rH IL-2) represented about 20% of the total E. coli protein and the rH IL-2 produced in E. coli was biologically active as demonstrated by the growth promoting effect on an IL-2 dependent cell line.Il6 The rH IL2 was purified with ultra purity by methods involving isolation of inclusion body, urea extraction, solubilization, and gel filtration ~hromatography,"~ and the purity was recognized by the Food and Drug Administration (FDA) of U.S.A. I n vitro augmentation of natural killer cell activity and mixed lymphocyte reaction assay were shown to be comparable with other rH IL-2 from different manufacturers and Iaboratories of other countries."' The clinical potential of rH IL-2 is going to be evaluated for Korean patients. The mature human immune interferon (IFN-y) gene was cloned as a cDNA form into E. coli high expression vector constructed by using tac promoter and rrmBT IT2 transcription terminator r e g i ~ n . ' ~The . ~ ~IFN-y produced by the E. coli harboring the gene showed cross-reactivitywith mouse anti-human IFN-y serum. INF-y could be purified by immunoabsorbant chromatography technique. The N-terminal sequence of purified INF-y was exactly identical to that of natural leukocyte INF up to 30 amino acid residue except the 1st and 29th unidentified amino acid residues. Human insulin A and B chain genes were chemically synthesized and cloned separately into E. coli using pUC19 vector, and the activity of the insulin synthesized by artificial genes is under investigation.120The structural sequences of human growth hormone (hGH), human chorionic somatomammotropin (hCS)-A, and hCS-B genes were isolated from a partial human genomic DNA library of ~ B R 3 2 2The . ~ ~structural sequence of hGH gene was fused with mouse metallothionein-I (MT-I) promoter and the expression plasmid DNA was introduced into mouse L-cells, where the gene was expressed under the control of the mouse MT-I promoter. When the MT-I promoter was induced by cadmium, the level of hGH was increased severalfold. The bovine growth hormone (bGH) gene was also cloned as a cDNA and the gene could be expressed in E. ~ o l i . ' ~ Production of several amino acids such as L-phenylalanine, L-lysine and L-tryptophan has been attempted by the recombinant DNA techniques in Korea. Among them, L-phenylalanine production was most successful. Phenylalanine synthesizing gene aroF and pheA was cloned in E. coli where the genes were overexpressed with the yield of phenylalanine from 13 g/l up to 43.7 g/l. This technique was developed by Miwon and the amino acid is going to be the first amino acid in the world sold in market produced by the recombinant DNA techniques. Several new restriction endonucleases, ZanI,'22BmaI,'23and C t h P were purified from Zymomom anaerobia (NCI B8227), Bacillus macerans ATCC8244, and Clostridium thermocellum ATCC20405, respectively. The recognition sequence of ZanI 1990

51

Critical Reviews In was determined to be 5'-CC f (AT)GG-3', the same sequence as RstM and EwRII, that of BmaI was 5'-CGAT t TCG-3' as isoschizomer of PvuIl and that of CthI was 5'-TGATCA3' as isoschizomer of Bcfl.


IV. PLANT BIOTECHNOLOGY The last decades have been extremely exciting for the plant breeders who have made it possible for the main crop, paddy rice, to be self-sufficient in Korea. The yield of paddy rice per unit area is known to be the highest in the world at about 4.48 M/T/ha. But limitation has been confronted for the more improvement of yield and quality of crops by the conventional breeding method because the pollination is the way to produce seeds only between the sexually compatible species. New biotechnologies like recombinant DNA techniques, cell culture, and somatic cell fusion are being applied to plant science in Korea because they have potentials to further increase crop yields, to decrease fertilizer requirement, and to manage weeds and pests without a great dependency on dangerous chemicals. Until the last 3 or 4 years, there was little interest in the basic plant science, plant molecular biology, or plant biochemistry. Even though a couple of government-supported research institutes including the Agricultural Research Institute have begun several projects on plants, the funding level was quite low comparing the granting programs in bacterial and animal including human basic sciences. But changes have started to occur in the last couple of years in Korea like other developed countries. A greater number of creative scientists from a variety of disciplines are beginning to study plant science and dedicated people are looking for training in the field. Not only the government-supported research institute but also private industries have become interested in plant biotechnology because plants naturally produce many valuable pharmaceuticals, coloring agents, flavoring agents, and pesticides. Several industries are placing a great deal of effort into manipulating the plants so that the product of interest is produced in far greater quantities. The research progress in plant gene manipulation, cell culture, cell fusion, artificial seed production, F, - hybrid seed production, secondary metabolite production by cell culture, and biological nitrogen fixation is reviewed in this article.

A. Cell Culture and Fusion Plant cell culture offers many exciting possibilities since it is possible to grow plants from a single cell. This technology has been used with several plants and it is just a matter of time before all major crops will be amenable to plant regeneration from single cells. The isolation of viable protoplasts and in v i m culture of the cells as callus form have been successful on a broad spectrum of plant species belonging to Brasiaceae, Solanaceae, Cruciferae, Umbelliferae, Leguminosacease, and Gramiaceae, but 52

the species of plants which could be regenerated to entire plant from the isolated protoplast were limited (Table 1 I ) . The successful isolation of viable protoplast, which is critical to cell culture, depends on the species of plants, parts of organs from which protoplasts isolated, and concentration of cell wall degrading enzymes. The optimum conditions for the isolation of protoplast, callus formation, and entire plant regeneration are varied from species to species. Mesophyll of young developed leaves are most frequently used as the source of protoplast but in some species it is necessary to use other organs like cotyledon or in v i m cultured cells for obtaining protoplast able to proliferate into colonies and ultimately to regenerate into plant. In order to obtain a useful somaclonal variants having better characteristics for crop improvement, selection of the spontaneous somaclonal variants due to chromosome instability of the cultured plant cells or induced mutants obtained by the treatment of chemical mutagens such as NaN, and N-methylN'-nitro-N-nitrosoguanidineduring the cell culture have been attempted but the number of valuable variants obtained from protoplast culture are limited in the crop plants except several ornamentals.

Table 11 Protoplast Culture Response of Various Plant Species Species Amaranthus tricolor Brassica napus Glycine spp. Hibiscus syriacus H . rosusiensis Hordeum vulgare

Protoplast source Cotyledon Mesophyll Cotyledon Callus

Mesophyll, cotyledon Cotyledon hypocoLycopersicon esculentum X L. pimpinelifohm cv. tyl, stem Lycopersicon esculentum, Sal- Mesophyll anum mebngena Nicotianu sanderue Mesophyll Oryza sativa Anther-derived callus Oryza sativa Suspension cells P a m Rinseng Suspension cells Petunia hybrida Mesophyll Populus nigru x P . Mesophy 11 muximowiczii Robina pseudoacacia Callus Solanum melongena Mesophyll Solanum nigrum Mesophyll Solanum tuberosum cv. Duezi Mesophyll Solanum tuberosum cv. Mesophyll Daejima Trifolium repens Mesophyll

Response

Ref.

Callus Shwt Rwt Callus

12s 126 127 128 129

Callus

I30

Callus

131

Shoot Shoot

I32 133

Cell division Cell colony Shoot Shoot

134 135

Cell division Shoot Shoot Cell division Callus

138 139 139 140 I25

Cell division

141

I36

137

A variety of approaches have been made to get stress resistant crops like salt-tolerant r i ~ e , ' ~ *cold . ' ~ ~tolerant rice,'"*'45 or acid resistant crops,'46 through protoplast culture, but the results obtained are not satisfactory so far.

Volume 10, Issue 1


Biotechnology In contrast to protoplast culture, tissue or organ culture to propagate a wide range of ornamental including orchids, especially oriental orchids, ferns, and lilies have been successful in commercial application. Many commercial laboratories including small horticulture farmers are routinely using this technique with commercial competitiveness in Korea. Pomato was an exciting word for the researchers working on plant cell fusion. Plant protoplasts are negatively charged to prevent the resulting repulsive forces. Rapid modification of the ionic environment of protoplasts by Ca'' mixed with 0.4 M mannitol in a solution of high pH(10.5) has led to cytoplasmic fusion of tobacco protoplast. The combination of polyethylene glycol, powerful aggregator of protoplasts, with high Ca2+ and high pH has been found to be more efficient. In order to obtain somatic hybrid by protoplast fusion, a dozen fusion combinations between genus or species of plants were fused (Table 12) but only a few intergeneric and interspecific somatic cells could be successfully fused and a part of them regenerated to entire plants. The regenerated plant was obtained from the interspecific somatic hybrids between Brassica napus B . oleracea126and Nicotianu plumbaginifolia N . sylvest r i ~ , ~and ~ Ofrom the intergeneric cell fusants between Petunia hybrida Solanum n i g r u ~ nand ' ~ ~Petunia hybrida + Solanum melongem."'

+

+

+

Table 12 Shoot and Callus Formation from Fusion Product Cultures in Various Fusion Combinations Cell fusants

+

Brassica napus B . campestris B. oleraceu B . napus Lycopersicon esculentum Solanum nigrum L. esculentum iDacus carota Nicotiana qlauca N. tabacum N . plumbaginifolia iN . sylvestris Nostoc muscorum Oryza sativa Petunia hybrida Solarum nigrum S . melongena P . hybrida Robinia pseudoacacia Lespedeza bicolor Solarum tuberosum iAmaranthus tricolor S . nigrum Dacus carota

+

+

+

Shoot

+

+

+

Response

+

+

Shoot

Shoot Shoot Callus

Ref. I47 126 148 148 149 150 151 152 153 138 125 148

B. Artificial Seed Production Since ReinerP4 and Steward et al.'55 induced somatic embryos, artificial embryos, from root tissue of carrot, research on somatic embryogenesis has been carried out mostly in the Umbelliferae, Rutaceae, and Solanaceae. The Murashige's proposal in 1977156that somatic embryos can be incapsulated to give birth to artificial seeds was not accepted until research on production of artificial seeds began in the 1980s. Plant Genetics, a venture company in the U.S. led by Redenba~gh'~'-'~' used natural hydrogels such as alginic acid for the capsule of somatic embryos. The other artificial seed

producing techniques are the use of polyoxyethylene oxide which is being developed by the Janick group of Purdue University, the fluid drilling technique which is attempted by the Cantliffe group of the University of Florida,'62 and the use of desiccated somatic embryos by Gray et al. of the University of Florida. The Janick group put small clumps of somatic embryos of carrot into polyethylene oxide and dehydrated them. When the water was resupplied, the somatic embryos were to germinate in vitro. The Cantliffe group is attempting to mix somatic embryos in place of seeds with gel and sow them directly to the field. Despite of their advantages of the methods other than the use of alginate capsule, they are significantly lacking the similarities from the true seeds, compared to the one somatic embryo in one capsule method used by Plant Genetics, Inc. These artificial seeds can be utilized in the vegetatively propagated crops such as sweet potato, potato and garlic, and high yield F, hybrid of corn and other major crops which should be crossed annually requiring much labor, time, and field. Meanwhile, the reasons that artificial seeds are frequently discussed in relation to genetically engineered plants are as follows. First, plant that is newly created by the recombinant DNA technique can be rapidly propagated in the artificial seed form in a large quantity. Second, the foreign genes that are put into plant genome are usually so unstable that they might be lost upon seed set. However, chances of losing the genes for useful characters are blocked by artificial seeds because there is no meiosis in the process. Third, even if the genes for useful characters can go through meiosis stably, they are scattered among the progeny and the scattering is prevented by artificial seed propagation. In Korea, one of the leading groups in this field is the plant cell culture laboratory, Genetic Engineering Center, KAIST, led by Liu. Liu et al.lM-IMsuccessfully produced artificial seed from somatic embryo of carrot (Duucus carota L. cv. Hongshimochon) with alginate capsule (Figure 1). They could dehydrate the alginate capsules containing carrot somatic embryos to the level of about 80% water loss with more than 90% of in vitro germinating rate. This germinating rate in vitro is conspicuously higher compared with those of Kitto and J a n i ~ k ' ~ ' and -'~~ with the system of Gray et al.163 The improvement of the germinating rate of the dry type artificial seed was achieved by supplementation of abscisic acid (-A) and or L-proline to the somatic embryo producing media. Many species of plant are known to accumulate L-proline under osmotic stress,'69 and it was reported that L-proline assists the friable embryogenic callus induction and the development of somatic embryo in maize.'70 However, it has not been reported that L-proline is used for increasing the tolerance of somatic embryos to dehydration prior to the study of Liu et al.1M-166 They also found that L-proline has a synergetic effect 1990

53

Critical Reviews In


In addition to these, when this dry type of artificial seeds were powder coated with a fungicide, Benlate, and left uncovered at room temperature for a few months, appreciable contamination was not observed with naked eyes. Some promising results for the production of artificial seeds from the somatic embryos of potato, sweet potato, and garlic are expected in the near future by application of the similar techniques (Liu et al., personal communication).

FIGURE 1. Hydrated and dehydrated carrot artificial seeds of alginate capsuleIw (A) freshly encapsulated somatic embryos, (B) gemination of hydrated artificial seeds, (C) dehydrated artificial seeds, (D) reimbibed artificial seeds, (E) germination of reimbibed artificial seeds.

with ABA in increasing the tolerance of carrot somatic embryos to dehydration. The hydrated artificial seeds by Redenbaugh et al. 1s8-161 and the fluid drilling technique for somatic embryos by Cantliffe et al.162have a faint analogy to the true seeds because they do not have the advantage of convenient storage, transportation, and handling of germplasm which the true seeds offer. The desiccated wafer system of Kitto and Jani~k'~'-'~* supplements the drawback of the previous two methods because somatic embryos are in a dry state. But because a few or a few tens of somatic embryos are put into one wafer, and the surviving rate even in vitro is very low, it cannot be competed with a true seed which has one embryo in one unit and has a considerably high tolerance to desiccation. On the other hand, the dried somatic embryo method of Gray et a1.'63made progress in approaching the dry state of the true seeds, but the naked somatic embryo does not have devices equivalent to the testa and endosperm. The latter is required for supplying nutrients during germination and early growth, especially in albuminous crops. In contrast, the dry type artificial seed developed by Liu et al. IM-16.5 has greater analogy to the true seed than any other types reported so far due to the following reasons. The artificial seed has one embryo per one unit, is dehydrated, has the protective device like testa and nutrients supplying mechanism during germination, does not germinate during storage under dry condition, but does germinate when water supplies. 54

C. Production of Secondary Metabolites by Plant Cell and Tissue Culture Plants naturally produce many phytochemicals that are Valuable pharmaceuticals, coloring agents, and flavoring agents. Some complex chemical structures of many compounds such as glycosides can be produced more easily and economically by plants rather than by organic synthesis. The market volume for some plant products are estimated over 100 million dollars annually worldwide. For the production of the plant products, tissue or cell culture appears to be an attractive method for a constant supply of homogenous material. Moreover, with in vitro cell culture, it may be possible to produce new active compounds or to convert low value substrates into high value compounds by biotransformation. Since Routien and Nickell"' took out a patent on the production of natural substances by plant tissue culture on a large scale in 1956 and in 1983 Mitsui Petrochemicals Industries in Japan successfully produced and commercialized shikomin from cell suspension cultures of Lithospermum erythrorbizon, several compounds such as anthraquinones, berberine, caffeine, rosmarinic acid, etc., are being produced by plant tissue culture with a yield higher than that of the intact plant.17* Recently in Korea, Korea Tobacco and Ginseng Research Institute (KTGRE)and several industries have started on this project for the production of Panax ginseng saponin and some other pharmaceuticals. Korean Panar ginseng root is very famous and well known worldwide because of its pharmaceutical activities to humans. Several kinds of ginseng drinks and tea made of ginseng root extracts are very popular not only in the Korean market but also in some Asian countries including Japan. But the ginseng root is quite expensive because it takes 4 to 6 years to harvest after sowing and also the plant cannot be cultivated in the same soil consecutively. The production of crude ginseng saponins by cell suspension culture of ginseng roots was succeeded in KTGRE and Japan. The problem in industrial application of the cell suspension culture is the scaling up of laboratory method to large-scale fermentation, and instability of plant cell lines because of genetic and/or epigenetic modification or asynchronousbiochemical differentiation which results in changing the bioreactive compounds compared to those from real plants. There are some limitation^'^^"^^ to obtain published references related to this field in Korea but it is obvious that sig-

Volume 10, Issue 1

Biotechnology


nificant research progress in this field is being achieved in private industry and government-supported research institutes.

D. Plant Gene Manipulation Plant improvement by the new molecular and cellular genetics techniques can be characterized by identification, chemical description, and cloning of desired genes in the laboratory, and ultimate insertion of desired genes into appropriate plants. Plant gene manipulation research using recombinant DNA techniques have not progressed much yet in Korea. A few plant genes have been cloned as a cDNA or genomic form. The glycinin gene, the most abundant storage protein of soybean (Glycine mar) was cloned as a cDNA clone by Kim et aLg3and characterization of the cloned gene is in progress. Proteinase inhibitor I and 11, a powerful inhibitor of serine endopeptidase such as trypsin and chymotrypsin, gene from potato (Solanum tuberosum) was screened from the EcoRl genomic library and its base sequence was compared with the gene from tomato.85 Chitinme, which catalyzes the hydrolysis of 1,4-N-acetyl glucosamine linkage of the fungal cell wall polymer chitin and serves as a component of the inducible defense of plants, gene cDNA clones were isolated from the cDNA library synthesized by poly(A) mRNA isolated from the ethepon treated leaves of pea by Kim et al.84They could transfer the cDNA of chitinase gene from pea leaves into tobacco leaf disks via a binary Ti-plasmid vector system and transgenic plant were regenerated. E. coli thioredoxin gene could be expressed in tobacco cell (N. tabacum cv Xunthi) and the transformed cell could be regenerated to the whole plant.'75 These are the only reports published in Korea which introduce foreign genes into other plant by plasmid vector and regenerate the transgenic cells into plant. Several other approaches than plasmid vector mediated transformation of plant cell such as microinjection and cocultivation techniques have been applied to transform plant cells. 176 Cho et al.'76 and Kang et a1.177.178 could transform radish by microinjection of the total DNA extract of Altari radish leaves into the ovary of Kungzung radish when the recipient was selfpollinated. Seven transformants out of 2760 plants were obtained when the seeds harvested from the DNA injected Kungzung radish were grown. The characteristics in the transformants were segregated to the ratio 3: 1 in the next generation as if the transformants were the hybrid between Altari and Kungzung radish. Cocultivation of immature embryo of Kungzung radish with the DNA prepared from Altari radish leaves could produce some transformants.177*178Production of genetic variants by microinjection and cocultivation techniques implies that such techniques could be a way to bypass the vector system in transformation studies.

+

E. F,-Hybrid Seed Production Hybrid vigor has made important contributions to the

development of crop production. Most of the research work has been concentrated into hybrid ~-ice,'~O-'~~ heat,'^^-'^^ and other crop^.'^^,'*^ Recently many seed companies, Rohm and Haas, Northup King, Cargil, Hybrid Teck, and de Pont have competitively invested a lot of funds for the modernization of technology. The traditional method for hybrid seed production is based on producing hybrid seeds through three lines, that is, male sterile, maintainer, and restorer. The use of chemical male gametocide to cause an artificial, nongenetical male sterile parent to produce hybrid seeds is another approach with great promise in utilizing hybrid vigor. This approach is called a two-line method. The mechanism of chemical emasulation is dependent on the difference in tolerance of male gametocide dosage between the pistil and the stamen. This means the pistil suffers from damage by the male gametocide. But under a certain dosage range the pistil is more tolerant of male gametocide than the stamen. With a lower dosage of male gametocide, the pistil can still live but the stamen will be severely damaged and lose its activity. If the dosage of male gametocide is too high, the pistil will also be severely damaged, resulting in remarkable reductions in outcrossing rate and hybrid seed yield. If the dosage of male gametocide is too low, the stamen will be tolerant of the damage and keep its activity, resulting in self pollination and failure of hybrid seed production. The critical range of male gametocide dosage is known to differ with variety, developmental stage, nutrient status, ecological conditions, and others. This method has advantages both in its scientific and economic significance. The two-line method, in which a chemical treatment is capabIe of temporarily making a bisexual plant male sterile, would have great potential in the commercial production of hybrid seed. The primary advantage of this approach is that a sterile line is not needed and that it is free of the unfavorable characteristics of a particular sterile line, such as unsusceptibility to disease or poor quality. In theory, any desirable variety can be used as the female parent to cross with any male parent. The possibility of the appearance of ideal combinations with good quality, high yield, and multiresistance will greatly increase. lS9 The approach makes breeding time greatly shortened. Because there is no segregation in the F,, as soon as hybridization in remote cultivars is successful and the F, is ideal, it can be used in crop production. If the parents of the F, combination are very similar in plant height and growth period, the seeds from the F, plants can be grown with certain hybrid vigor in yield. The main barriers we have to overcome are (1) discovery of effective and practical chemical male gametocide, (2) hybrid seed production on a large scale, and (3) yield increase of chemically emasculating hybrid seed crop by full exploitation of hybrid vigor. In Korea, hybrid seed production in cereal crops has been out of focus until Lee et al.l9O verified a possible exploitation of hybrid vigor in rice. Heu and ChaeI9' used FW-450(sodium 2,3-dichloro isobutyrate), MH-30(maleic hydrazide-30) and

1990

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__

Critical Reviews In

TIBA (2,3,5-triiodobenzoic acid) and proved unsuitable for inducing male sterility in rice. The first report on successful male sterility in cereal crop was made by with ethrel (2-chloroethylphosphonicacid, CICH, HPO,) in barley and wheat. He found that ethrel induced practical level of male sterility in barley but not in wheat. Later on the effects of chemicals on male sterility were reported in various crops as shown in Table 13. We can conclude that the effects of the chemicals used as gametocide were specific to crops, cultivars, and growth stages of the parents. Therefore, it is necessary to find out the specific condition for inducing male sterility without serious phytotoxicity. The concentration and spraying method would be critical in most cases. Recently, the second phase of the research is being initiated by Choe et al. 191.194 with the development of the hybrid-seed production system. They attempt to assess the effect of parent arrangement and outcross rate in different combinations, as well as extra yield potential in addition to finding out a possible relationship between the effects of chemicals and the alteration in leaf protein properties. They recognize that planting spaces

of pollinators are of great importance in order to obtain high outcross rates in barley and wheat. Conclusively, research on hybrid-seed production by chemical emasculation in Korea is still rather primitive; however, a great deal of interest is given to this area in order not to be defeated in major international seed markets. Limited information and research personnel shortage hinder the development of the techniques.

F. Nitrogen Fixation The growth of plants, agricultural crops and forests, is largely depend on tremendous supply of nitrogen, either mechanically applied as ammonia, urea, or nitrate fertilizers, or naturally produced in the soil through microbial reduction of atmospheric nitrogen called biological nitrogen fixation. During the last decades, the increasing energy costs and development of the recombinant DNA technologies encouraged research on the biological nitrogen fixation to increase the available supply of naturally reduced nitrogen. Although most organisms including all the eukaryotes cannot assimilate atmospheric nitrogen, a limited number of prokar-

Table 13 Chemicals, Concentration, and Growth Stage at Treatment Male Sterility In Various Crops Crops

Barley

Rice

Chemicals

Concentration

Ethrel 1-2000 pprn Booting (with surfactant) “Reno” Ethrel 4000--8000 ppm 4000--8000 ppm NH-30 FW-450

0.2%

MH-30

0.05%

MH-30

56

2-3 weeks before heading

4000--8000 rpm

Booting

Earformation Booting Booting

TIBA Ethrel NH-30

Wheat

Growth stage of treatment

ppm Preboot

Ethrel

1000-2000

Ethrel

4000-8000 pprn Booting

Volume 10,issue 1

Response

Ref.

Effective in barley 4 U % sterility Less effective Cultivar specific Leaf discoloration

192

Inhibited heading Highly effective in indicatype (80%) less effective in japonicatYPe (40-70%) Not effective Less effective Highly effective (53-90%) Less effective boot, after boot Effective (4G-70%)

191

193 154 191

193, 194

191 193, 194 193, 194

192

193, 194


Biotechnology yotes are able to reduce nitrogen gas to ammonia. The prokaryotes capable of nitrogen fixation, diazotrophs, can be classified into three categories, that is, free living, associative, and symbiotic nitrogen fixers. 195 Free living nitrogen fixing bacteria which fix nitrogen in the state of not intimately associated with specific plants include members of Klebsiella, Azotobacter, and Clostridium. The associative nitrogen fixer like Azospirillum fix nitrogen in close association with roots of cereal^'^^,^^^ and tropical grasses.I9’ The third group of nitrogen fixers are Rhizobium which nodulate leguminous plant and only fix nitrogen when they are symbiotically associated with the plant, 199*200 Anabaena which fixes nitrogen within the leaf pores of the water fern Azolla201and Frankiu which nodulate alder trees.202 The enzyme complex for nitrogen fixation, the 17 contiguous nflnitrogen fixation) gene products organized in seven operon, has been studied in detail in K . pneumoniue.195The knowledge obtained from the study of this organism is helping to understand the more complex but agronomically important associative processes of Azospirillum and symbiotic processes of Rhizobium. The associative nitrogen fixer, Azospirillum spp., were reported to contribute to increased yield of cereals or grasses not only due to nitrogen fixationzo3but also production of plant growth promoting substances like indole-3-acetic acid (IAA).ZW,~O~ The symbiotic nitrogen fixation is carried out within highly differentiated root nodules formed by interactions of Rhizobium with plants of the legumes (soybean, alfalfa, peanut, clover, pea, bean, etc.) enabling these plants to grow without or with little addition of nitrogen fertilizers. In Korea, the associative nitrogen fixer with paddy rice, Azospirillum spp., has attracted considerable interest because paddy rice is the main crop which dominates over 50% of the total agricultural production. Several A. lipoferum strains which fix nitrogen effectively with the association of Korean paddy rice cultivars were isolated from Korean paddy roots and soils. Genetic modification of the isolated Azospirillum strains was attempted to overcome the ammonia-regulation, repression of nifgene expression by the fixed nitrogen, and to increase production of the phytohormone, IAA.’” The ammonia-regulation was partially overcome by the introduction and constitutive expression of the nifA gene, an activator gene for whole nif gene promoters, of K . pneumoniae in the A . lipoferum host.’” The IAA is synthesized from tryptophan via indole-3-pyruvate by the key enzyme, tryptophan aminotransferase. The wild type Azospirillum produced trace amount of IAA in the media without supplementation of tryptophan because of the limited amount of tryptophan synthesis. The IAA synthesis in Azospirillum could be increased by the introduction of trp operon from E . coli which has tryptophan feedback insensitive mutation.207In a pot experiment,

about 15 to 20% of yield increase of paddy rice could be obtained by the inoculation of the genetically engineered A. lipoferum strain for the overproduction of IAA.’07 Another trend of research is being carried out with the possibility that increased symbiotic nitrogen fixation will increase yield of leguminous plants, or that additional plant varieties could be made capable of carrying out nitrogen fixation. The effective symbiotic nitrogen fixation of Rhizobium legume depends on genetic information present in both the bacterial and host plant genomes. The potential for improving capacity of symbiotic nitrogen fixation by gene manipulation of Rhizobium seems to be much higher than that obtainable through genetic engineering of the host plant because of the complexity of the host plant genes involved in symbiosis. Several Rhizobium strains which are more effective to Korean soybean cultivars were isolated208.2w and inoculum for soybean produced with the isolated Bradyrhizobium japonicurn strain is going to appear on the market this year. Also, several Rhizobium mutants having better characteristics of nitrogen fixation were obtained by the induced mutagenesis although the mutant strains could not be developed as an inoculum due to ecological incompetitiveness with the indigenous wild type Rhizobium distributed in the The possibility for the introduction of bacterial nifgenes into plants like cereals or creation of a new cereal symbiosis like Rhizobium legume symbiosis by genetic engineering is being explored in several laboratories in Korea like leading research groups in this field in other countries, but it seems to have numerous obstacles to overcome.

V. MONOCLONAL ANTIBODIES The long-lived cloned cell line can be prepared in vitro by chemically mediated fusion or hybridization of lymphocytes from immunized mice and a mouse tumor (myeloma) cell line. The hybrid cells have antibody-producing capability from the lymphocyte and ability to grow permanently in culture from the myeloma cells. The cells have been used to prepare antibodies against many viruses, bacteria, fungi, and parasites. The animal cell hybridization and monoclonal antibody preparation techniques have been introduced to Korea in the early 1980s and antibodies of diagnostic potential have been prepared in research laboratories against a variety of viruses, bacteria, fungi, and parasites, which cause a serious health problem for the humans and animals living in the Korean peninsula. The monoclonal antibodies which have been reported to be prepared up to 1988 are against the enterotoxins211-213 and pili2I4 of Escherichia coli, Salmonella typhimurium, 215 Vibrio vulnificus.2’6 Leptospira interrogans,217.218 Chlamydia trachomatis,2’9.220Rickettsia Rickettsia tsutsugamushi,**‘ Mycobacterium tuberculosis,z22~223 Herpes simplex virus,224 Marek’s disease Cytomegalovirus,226Hepatitis B vi1990

57


Critical Reviews In rus,227.2z8 Hantaan virus,229avian infectious laryngotracheitis virus (ILTV),230avian infectious bronchitis virus (IBV),231 Newcastle disease virus (NDV),232 transmissible gastroenteritis virus (TGEV),,,, bovine viral diarrhea virus (BVDV)?” Taenia soliuni of cysticerco~is,~~~ a-fetoprotein (AFP)2193236.237 of hepatoma, carcinoembryonic antigen (CEA)238of colon cancer, surface antigen of stomach cancer,239CALLA240.241 of acute lymphoblastic leukemia, cell surface antigen of human T lymphocyte ,242-244 estradiol,245.246 bovine immunoglobulin IgG 1, and abscissic acid.247 Ten leading causes of diseases from which Koreans are dying are a little different from those of the welfare societies of advanced western countries. They are cerebrovascular disease (1 st), hypertensive disease (2nd), cardiovascular disease (3rd), stomach cancer (4th), liver cirrhosis (5th), primary hepatocellular carcinoma (6th), traffic accidents (7th), other accidents (8th), tuberculosis (9th), and other chronic pulmonary diseases (10th).248Among these ten diseases, special attention will be paid to the viral hepatitis types B, tuberculosis, and a recent achievement on a Korean endemic disease. Viral hepatitis type B is a major public health threat in Korea. About 10% of the population carries the serological marker of hepatitis B virus (HBV), H B s A ~ . ’ ~ ~ The hepatotrophic virus not only produces acute hepatitis, but also leads to chronic liver diseases terminating in parenchymal liver cell failure, cirrhosis, and death. Currently, HBV vaccines as mentioned in the earlier part of this review are produced commercially in Korea by the recombinant DNA techniques and available for mass vaccination. Tuberculosis is the 9th killer in Korea. The number of patients with active tuberculosis is estimated to be 800,OOO (2.1% of the total population) .2so Mass screening and mass treatment are essential for obvious reasons. Unfortunately, mass screening by conventional tools of diagnosis, such as chest X-ray, sputum smear, and culture are evaluated neither effective nor efficient. The fact that more than 55% of the Korean patients do not know their diseases and therefore do not seek medical help may explain in part the inefficiency and ineffectiveness. Mantoux reaction is also ineffective because more than 95% of Koreans above the age of five are Mantoux-positive. With these regards, a more simple and sensitive test with a high accessibility, like the VDRL test for syphilis, might enhance the effectiveness of mass screening. Although many investigators have tried to develop such kinds of immunological tests for a long time since Robert Koch described the tuberculin reaction, the tests, without exception, resulted in poor sensitivity. With the monoclonal a n t i b ~ d y and ~ ~ ~recombinant ~~*~ DNA251 technology, the antigenic analysis of Mycobacterium tuberculosis at the epitope level is being analyzed for the development of serological diagnosis of active tuberculosis. Acute febrile illness of unknown etiologies is believed to have existed for a long time in rural areas of Korea. From the early 1970s, the endemic disease has been recognized as a

58

public health threat. Since then, the etiologic agents have been isolated and identified as Hantaan virus of hemorrhagic fever with renal syndrome, Leptospira interrogans of leptospirosis, Rickettsia tsutsugamushi of scrub typhus, and Rickettsia typhi of endemic typhus. These pathogens are serologically crossreactive within the related agents. Obviously, monoclonal antibody with desired specificity was required to identify and differentiate the organism.z21*z52 Some of the developed monoclonal antibodies are already in practical use for the diagnosis of microbial disease with immunofluorescence or radioimmunoassay system. It is believed that development of the monoclonal antibody techniques for the production of diagnostic reagents should lead to great improvement not only in accuracy but also in time for diagnosis of microbiological infection.

VI. ANIMAL EMBRYO MANIPULATION AND TRANSPLANTATION The technology of embryo manipulation and transplantation has been extensively studied and widely applied in the field of animal reproduction because of its industrial significance. Current applications of the techniques to animal reproduction include promotion of genetic improvement of domestic animals, rapid increment in the number of heads of new breed, acceleration of progeny test, shortening of generation interval, multiple fetation, identical twin production, long-term storage of gametes, production of chimeric or transgenic animals, and so on. With the advent of molecular biology and concurrent advances in animal cell culture, micromanipulation techniques are playing increasingly important roles in animal genetic engineering. In Korea, Goh et al.253firstly applied non-surgical embryo transfer technique to produce twin calves with Korean native cattle. Thereafter three groups of researchers have been successful in embryo transfer in cattle with fresh or frozen embryos.254-256 Progress has been made toward developing efficient superovulatory i n d u ~ t i o n , ~synchronization ~’-~~~ of estrus,2~~z62 and improving pregnancy rate following bovine embryo transfer .263-265 Goats are widely used as a model for cattle in various studies in Asia. Song and Park2“ investigated the induction and synchronization of estrus with prostaglandin F 2a in Korean native goats. They reported that following induced abortion, parturition and superovulation of Korean native goats, as other kinds of goats, showed high frequency of short estrus cycles, which were resulted from premature luteal regression and affected the quality of embryos. More effective synchronization of estrous cycle, better ovarian responses in ovulation and higher embryo recovery rate were achieved by primming with 10 mg of progesterone for each 10 d and followed 8 d after primming by injection of 20 mg of FSHp in decreasing dosesz6’and kids were delivered after embryo trar~splantation.~~~~~~~ Through the technique of embryo bisection, not only a significant increase in the number of embryos can be produced per donor collection,

Volume 10, Issue 1


Biotechnology Experimental methods have included centrifugation, sedibut also monozygotic twins, being genetically identical, are mentation, millipore filtration, electrophoresis, immunoselecbeneficially used as experimental animals in many types of tion, immunosedimentation, and alteration of pH. These biological investigation. In technique of splitting early staged methods were not proven practical. Recently, two methods of embryos, it involves the mechanical removal of zona pellucida, separating Xand Y-bearing spermatozoa have been developed separation of blastomeres, insertion of the separated blastowith a remarkable success rate. In the first method, the semen meres into evacuated zonae, embedding in agar, culture in was laid over bovine serum albumin in order to obtain high intermediate host, recovery of agar chips, removal of agar and proportions of Y-bearing spermatozoa. For the second method, retransfer to recipient animals. In this procedure, it requires the semen was passed through columns of Sephadex G 50 in the presence of an intermediate host and skillful techniques in order to increase the proportion of X-bearing spermatozoa. Lee manipulation. On the other hand, bisection of embryos at the et al.281applied Sephadex gel filtration technique in combistage of compacted morula to hatched blastocyst is relatively nation with density gradient centrifugation to the separation of simple and the pregnancy rate obtained is similar to that which X- and Y-bearing bull spermatozoa. Following centrifugation is usually obtained after transfer of intact embryos in the of discontinuous percoll density gradient, the higher motility mouse.270~271 It is generally believed that the presence of the index was obtained at the 5th fraction but the percentage of zona pellucida or any substitute protective layer is not essential B-body bearing spermatozoa was decreased a little from 39.7 for the successful development of demi-embryos at later than to 25.6%.Sephadex gel filtration did not much affect the mothe eight-cell stage. Also, no difference was found in the surtility of spermatozoa but prominantly decreased the rate of Bvival rate of demi-embryos produced from morulae or blasbody bearing spermatozoa to 12.0%. For sexing embryos, the tocysts. Park et al.270have attempted a number of different antibodies to H-Y antigens are usually made in rodents, and procedures for the improvement of viability of embryos after also the monoclonal antibodies to the antigens were probisection. The factors which may affect development of the duced.282The embryos are exposed to anti- H-Y antibodies, bisected embryos, i.e., the developmental stage of embryos which bind to the male embryos. Two methods are commonly and in vitro culture of the bisected embryos, were investigated used to detect the bound antibodies. The first method involves and further the technique was applied to the production monadding complement which results in the death of male embryos. ozygotic twins in goats. H ~ a n applied g ~ ~ this ~ technique to the KO et al.283and Han et aLZe4tested the effect of H-Y antibody induction of twinning in cattle. The development of reliable and complement on in v i m development of mouse embryos. methods for long-term preservation of embryos of the large Of the embryos treated with H-Y antigen and complement domestic species would greatly facilitate a more widespread 55.6%of embryos developed to blastocyst and 44.4%of them application of embryo transplantation in the breeding of farm were destroyed or arrested. Yang and Kimzs5also discovered animals. In addition to this, the cryopreservation of embryos that almost 50% of the embryos developed. Kimza6investigated enables saving the embryos exceeded recipient availability, the sex ratio of progeny after treatment of protein column and saving the expense for importing and exporting livestocks, and H-Y antibody in ewes. The male to female ratio of kids profurther preservation of breeds and genetic information. The duced after culturing the blastocysts in medium containing Hsurvival and normal development of mouse and rat embryos Y antibodies and compliments treated with spleen and testis after freezing and storage in liquid nitrogen have been achieved was 21.0:79.0 and 17.4:82.6, respectively. The more practical by techniques involving treatment of the embryos with media method is the second antibody method. This consists of procontaining dimethylsulfoxide or glycerol combined with relatively slow rates of freezing and t h a ~ i n g . The ~ ~ similar ~ - ~ ~ ~ ducing fluorescent-labeled antibody to the first antibody so that the male embryos will fluoresce in the light. Yang et al.287 methods were applied successfully to freezing bovine emmeasured the sex ratio of mouse embryos treated with H-Y b r y o ~In. ~cattle, ~ ~ when the frozen embryos were transferred antiserum and FITC anti-mouse-IgG by indirect immunofluto recipients whose estrus were artificially induced by PGF orescence of sexing embryos in the technique was around 77%. 2a,the pregnancy rate was 66%and that percentage was almost Goh et aLZ8' applied this technique to the sexing of bovine same as the rate with the fresh embryos.277The first birth of embryos prior to transfer to recipient. It has been demonstrated one calf following transplantation of frozen embryos imported that transgenic offsprings can be produced by injecting cloned from America was produced in Korea in 1984 by Seok et al.278 genes into nuclei of mammalian embryos and cloned offsprings The more simplified procedures of freezing and thawing of can be produced by nuclear transplantation. A prerequisite for embryos using a glycerol diluent containing sucrose are under injecting foreign genes into a nucleus or injecting a nucleus i n v e s t i g a t i ~ n .For ~ ~the ~ * determination ~~~ and selection of preinto the mammalian embryos is a method of visualization of natal sex of animals and humans, two categories of experiments the nucleus. Although the pronuclei or nuclei are easily viswere conducted. One is separation of X- and Y-bearing sperualized in mouse and rabbit embryos, in pig and sheep embryos matozoa and the other is sexing embryos. Numerous attempts they are obscured by large lipid granules in the cytoplasm. have been made to separate X- and Y-chromatin-bearing However, three methods have been used to locate pronuclei or spermatozoa.

1990

59


Critical Reviews In nuclei: ( 1 ) labeling nuclei with a vital fluorescent dye, but with this method the exposure of embryos to ultraviolet light is very damaging to the embryo, (2) using interference contrast microscopy, or (3) combination with centrifugation of the embryos to clear cytoplasm. Chung et a1.289and Lee et carried out an experiment on nuclear transplantation in mouse embryos. They removed male pronuclei by inserting a micropipette through the zona pellucida and a male pronucleus was inserted into an enucleated embryo by cell fusion mediated by inactivated Sendi virus. As a result, of 194 embryos nuclear transplanted, 126 (64.9%) embryos were fused and 68 (35.1%) embryos developed to blastocyst in vitro. Recently, the structural gene of human growth hormones (hGH) could be transferred to mouse egg cell by microinjection of the hGH fused with mouse MT-I promoter and transgenic mice having 1.5 to 2.0 times heavier weight than control was obtained.2q’In the future, these techniques will probably be of great importance to farm animal production through their use in cloning, gene transfer, and fertilization.

VII. PROTEIN ENGINEERING Organisms can synthesize proteins corresponding to the instruction furnished by a gene, which may be obtained from another life form or artificially synthesized gene. Modification of many different properties of protein, protein engineering, is now possible by combining the information on protein structure obtained from X-ray crystallography and computer modeling with the chemical synthesis of DNA. Recent advances in chemical synthesis of DNA now permit virtually unlimited genetic modification to create novel proteins not found in nature. Due to these prospects or potentials, several research groups in Korea are interested in protein engineering research, one of which is protein engineering research group in Genetic Engineering Research Center, KAIST led by Lee.’20,292They have synthesized a variety of insulin genes having one or several different codons from human insulin genes and are analyzing the activity of the insulins synthesized by artificial genes, although the complete results are not published yet. Other research on protein engineering by site-directed mutagenesis and chemical modification of protein2” has been attempted but the research on protein engineering in Korea is just beginning.

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Biotechnology from Escherichia adecarboxylare in Zymomonas mobilis, Proc. Mol. Biol. Genet. Korea, 3(1), 371, 1986. 24. Kho, Y. H., Cloning of glucose isomerase gene from Streptomyces phaeochromogenes in Streptomyces lividans, Korean J . Appl. Microbiol. Bioeng., 12, 253, 1984. 25. Park, H. D., Hong, S. D., Park, W., Park, J. S., Park, W. C., and Rhee, I. K., Development of alcohol-fermentingyeasts from Dxylose isomerase gene: overproduction of D-xylose isomerase in Escherichia coli. Proc. Mol. Biol. Genet. Korea, 2, 253, 1987. 26. Rhee, I. K., Park, H. D., Bae, S. C., Park, W. C., Jo, J. K., and Seu, J. H., Development of alcohol-fermentingyeasts from D-xylose by the cloning isomerase gene: Cloning of D-xylose isomerase gene in E. coli, Proc. Mol. Biol. Genet. Korea, 1 , 139, 1986. 27. Yu, J. H., Chung, K. S., Kong, I. S., and Lee, J. K., Cloning of the pullulanase Gene from Klebsiella pneumoniae NEB-320 in Escherichia coli. Korean J . Appl. Microbiol. Bioeng., 15, 141, 1988. 28. Yu, J. H., Chung, K. S., Kang, I. S., and Lee, J. K., Restriction mapping of cloned pullulanase gene and property of pullulanase produced in Escherichia coli (pYKL451 ) and Klebsiellapneumoniae NFB320, Korean J. Appl. Microbiol. Bioeng., 15, 436, 1987. 29. Chung, D. H., Shim, S. K., Maruyama, Y., and Chung, H. K., Molecular cloning and expression of raw-starch digesting gene produced by Bacillus s ~ . ,Proc. Mol. Biol. Genet. Korea, 3(1), 281, 1988. 30. Roh, Y. T., Jeong, B. C., and Lee, K. J., Molecular cloning of alkaline protease in Streptomyces spp. and optimization of fermentation: Role of alkaline protease in the cell differentiation, Proc. Mol. Biol. Genet. Korea, 3(1), 57, 1988. 31. Ahn, J. W., Oh, T. K., and Park, K. H., Molecular cloning and expression of alkalophilic bacteria protease gene in Bacillus sp. Purification and characterization of alkaline protease from a Corynebacterium sp., Proc. Mol. Biol. Genet. Korea, 1, 233, 1986. 32. Kim, I. C., Jang, S. Y., Lee, C. H.,Shin, H. K., and Park, K. H.,Molecular cloning and expression of alkalophilic bacteria protease gene in Bacillus sp., Proc. Mol. Biol. Genet. Korea. 3(2), 219, 1988. 33. Hong, S. D., Kim, 9. W., Kim, T. H., Park, S. H., and Lee, D. S., Cloning of the gene coding for 3-isopropylmalatedehydrogenase of Kluyveromyces fragilis and its expression in Saccharomyces cerevisiae and Escherichia coli, Proc. Mol. Biol. Genet. Korea. 3(1), 15, 1988. 34. Sun, J. M., Choi, W. K., Chun, S. B., and Kim, K. H., Cloning of Sacchuromyces cerevisiae glutamine synthetase gene. I. Isolation of glutarnineauxotrophic mutant, Proc. Mol. Biol. Genet. Korea, 3( I ) , 171, 1988. 35. Kim, E. K., and Lea, H. Z., Molecular cloning and restriction endonuclease mapping of homoserine dehydrogenase gene (HOM 6) in yeast Saccharomyces cerevisiae, Korean J . Microbiol., 24,357, 1986. 36. Kyoung, J. O., Choi, S. J., and Yang, C. H., Molecular cloning of hydrogenasegene in Escherichia coli, Korean Biochem. J . , 20, 129, 1987. 37. Choi, S. J. and Yang, C. H., Cloning and sequence analysis of E . coli hydrogenase genes, Proc. Mol. Biol. Genet. Korea, 3(2), 209, 1988. 38. Yu, J. H., Park, Y. S., Kim, J. M., Kong, I. S., and Chung, Y. J., Cloning of pectate lyase gene of alkali-tolerantBacillus sp. YA14 and its expression in Escherichia coli, Korean J . Appl. Microbiol. Bioeng., 16, 316, 1988. 39. Yoon, J. K., Choi, I. S., Huh, S. O., Chw, S. Y., and Park, S. D., Molecular cloning and characterization of RAD + gene required for excision of UV-damaged in Sacchuromyces cerevisiae, Korean J . Genet., 7, 97, 1985. 40.Song, Y. H. and Kang, H. S., The studies on the mechanism of RNA splicing in yeast molecular cloning of RNAl gene in yeast, Proc. Mol.

Biol. Genet. Korea, 1, 395, 1986. 41. Lee, H. S., Yoon, S. C., Lee, K. S., and Lee, C. W., Cloning and characterization of the ski2 gene of Saccharomyces cerevisiae, Proc. Mol. Biol. Genet. Korea, 3(1), 27, 1988. 42. Chung, D. H., Oshima, Y., and Nishiwaki, K., Studies on the his, gene of yeast his,-lacZ fusion and regulation in Sacchuromyces cerevisiae, J . Korean Agric. Chem., 28, 36, 1985. 43. Song, Y. H., Seo, S. Y., and Kang, H. S., Molecular cloning of the ADEl gene of the Saccharomyces cerevisiae, Korean Biochem. J . , 17. 399, 1984. 44.Choi, S. I. and Lea, H. Z., Molecular cloning and restriction analysis of aspartokinase gene (HOM3) in the yeast, Saccharomyces cerevisiae, Korean J . Microbiol., 26, 32, 1988. 45. Baik, Y. J., Chung, S. H., and Yang, C. H., Cloning of ada gene of E. coli K12 into E . coli ada mutants, Korean Biochem. J . , 19, 281, 1986. 46. Choe, S. Y.,Jung, Y. K., Joo, J. H.,and Park, S. D., Cloning and expression of DNA repair gene involved in adaptive response, Proc. Mol. Biol. Genet. Korea, 2, 41, 1987. 47. Lee, M. J. and Walk, J. R., Cloning and transcription of Escherichia coli cell division gene, sep, Korean J . Microbiol., 22, 235, 1984. 48. Huh, T. L., Kim, H. S., and Lee, S. Y., Cloning and expression of XP,-promoter containing recombinant trp gene in Escherichia coli, Korean Biochem. J . , 20, 91, 1987. 49. Chi, Y. T.,Kim, B. M., and Lee, S. Y., In vivo cloning of E . coli K12-trpL (Aatt) trpE gene using the plasmid RP4 :: Mucts, 61, Korean Biochem. J., 14, 266, 1984. 50. Park, W.,Song, B. H., and Hong, S. D., Cloning of acetyl CoA carboxylase (fabE) in Escherichia coli, Korean J . Appl. Microbiol. Bioeng., 14, 181, 1986. 51. Lee, Y. C. and Kang, H. S., Studies on the expression of tRNA (Asp) gene in Aspergillus nidulans, Proc. Mol. Biol. Genet. Korea, 2, 69, 1987. 52. Lee, Y.C. and Kang, H. S., Studies on the expression of tRNA(Asp) gene in Aspergillus nidulans, Proc. Mol. Biol. Genet. Korea, 3(1), 39, 1988. 53. Lee, B.J. and Kang, H. S., Studies on the organization and expression of tRNA(Arg) gene in Aspergillus nidulans. Proc. Mol. Biol. Genet. Korea. 1, 157, 1986. 54. Hong, Y. K., Cloning of 17s-ribosomal RNA gene from the hygromycin resistant Tetrahymena thermophila, Korean J . Appl. Microbiol. Bioeng., 14, 133, 1986. 55. Choi, Y. C., Kim, J. Y., Yu, M. H., and Han, M. H., Expression of a DNA sequence encoding for the pre-S2 region of hepatitis B virus in E. coli. Korean Biochem. J . , 19, 383, 1986. 56. Choi, Y. C., Kim, J. Y., Yu, M. H., and Han, M. H., Expression of hepatitis B virus surface antigen gene in E. coli, Korean Biochem. J . , 19, 377, 1986. 57. Kim, K. T., Song, K. B., Choi, Y. C., Rhee, S. K., and Han, M. H., Cloning of the structural gene for hepatitis B virus surface antigen into a yeast vector, Korean Biochem. J., 18, 122, 1985. 58. Kim, Y. S. and Kang, H. S., Cloning and expression of hepatitis B virus surface antigen gene, Korean Biochem. J . , 17, 70, 1984. 59. Kim, Y. W., Kim, K. S., Suh, Y. T.,and Gentaka, R. V., Cloning of reverse transcriptase gene of avian sarcoma virus, Proc. Mol. Biol. Genet. Korea. 2, 33, 1987. 60.Kim, Y.W.,Kim, K. S., Suh,Y. T.,and Guntaka, R. V., Cloning of reverse transcriptase gene of avian sarcoma viruses, J . Korean Agric. Chem., 31, 219, 1988. 61. Lee, J. S., Chung, S. H., and Sim, W. S., Cloning of the gene cluster from Klebsiella pneumoniae chromosomal DNA and expression in E. coli. Proc. Mol. Biol. Genet. Korea, 1, 383, 1986. 62. Chung, K. S., Lee, J. K., Mheen, T. I., Pyun, Y. R., and Yu, J. H., Cloning of nif genes from Enrerobacter agglomerans in Esch-

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Critical Reviews In erichia coli, Korean J . Appl. Microbiol. Bioeng., 15, 116, 1987. 63. Ayun, N. D. and Ryu, D. Y., Molecular cloning of the gene for Qacylamino-P-lactarnacylhydrolase from Acetobacter turbidans by immunochemical detection method, Korean J. Appl. Microbiol. Bioeng., 16, 363, 1988. 64. Lee, S. H., Paik, H. D., and Oh, Y. J., Molecular cloning of toxin gene from Bacillus thuringiemis var. kurstaki HD-I , Korean J. Appl. Microbiol. Bioeng., 16, 33, 1988. 65. Kim, S. H., Kim, Y. H., Park, E. W., and Kang, S. K., Cloning and expression of the Bacillus fhuringiensis TD- 1 crystal protein gene in Escherichia coli, Proc. Mol. Biol. Genet. Korea, 2, 83, 1987. 66. Kang, K. Y., Song, Y. H., and Cho, M. J., Cloning, cotransformation and expression of entomocidal toxin genes originated from B. thuringiensis supsp. kursfski and israelensis, Proc. Mol. Biol. Genet. Korea, 2, 91, 1987. 67. Kim, S. H., Park, E. W., and Kang, S. K., Cloning the crystal protein gene of B. fhuringiensis in E. coli and purification of B. thuringiensis RNA polymerase, Proc. Mol. Biol. Genet. Korea, 3(1), 237, 1988. 68. Kim, J. S., Lee, E. K., and Yang, C. H., Cloning of subtilisin carlsberg gene from Bacillus licheniformis, Korean Biockem. J . , 21, 176, 1988. 69. Jun, H. S., Kim, Y. S., Choi, K. R., and Rho, H. M., Cloning and expression of the BdiI methylase gene in E. coli. Korean J . Microbiol., 25, 40, 1987. Y. B., Oh, S. J., and Kang, M. S., Studies on 70. Kim, Y. C., MOO, the structure and expression of the penicillin G acylase gene. I. Cloning of the penicillin G acylase gene of E. coli ATCC 1 1 105, Korean J. Microbiol., 21, 95, 1983. 71. Roh, D. C., Kim, J. H., Park, S. K., Lee, J. W., and Byum, S. M., Molecular cloning of streptokinase gene from Streptokinase equisimilis and its expression in E. coli. Korean Biochem. J . , 19, 391, 1986. 72. Kwon, D. H., Kim, Y. S., and Byeon, W. H., Studies on antibiotics resistance gene in Staphylococcus aureus plasmid: cloning of cloramphenicol resistance determinant, Korean 1. Microbiol., 24, 341, 1986. 73. Kim. Y. S., Jeong, S. W., Kim, B. H., Lee, M. H., SUI, D. S., and Kang, H. S., Cloning of bovine growth hormone cDNA and its gene and expression of f f i H cDNA in E. coli, Korean Biochem. J . , 20, 67. 1987. 74. Uk,N. G.,Kim,Y. J.,Lee, K. K.,Han,M.H.,andKim,J.Y., Cloning of the structural sequences of human growth hormone gene family into plasmids and its utilization in transient expression systems, Korean Biochem. J . , 21, 226, 1988. 75. Yoo, J. O., Kim, S. K., and Chung, H. K., Molecular cloning of a cDNA sequence for bovine pepsinogen, Korean Biochem. J., 18, 240, 1985. 76 Bae, S. C., Rho, 0. K., and Lee, S. K., Regulation of LDH gene expression. I. Molecular cloning of rat liver full-length expression cDNA library and isolation of full-length LDH A-cDNA, Proc. Mol. Biol. Genet. Korea, 1, 165, 1986. 77. Suh, Y. H., Chun, Y. S., Kim, S. S., Kim, H. S., and Park, C. W.,Molecular cloning and nucleotide sequence of cDNA human epinephrine synthesizing enzyme (PNMT) and mRNA mapping in brain, Proc. Mol. Biol. Genet. Korea, 3(1), 93, 1988. 78. Kwack, K. B., Hyun, H. H., Kwon, B. S., and Kim, K. S., Highlevel expression of human immune interferon in Escherichia coli by using tac promoter, Korean Biochem. J . , 21, 53, 1988. 79. Koh, H. K., Hyun, H. H., Yoo, M. Y., Kwon, B. S., Tue, C. K., Moo,H. M., and Kim, K. S., cDNA cloning and expression of a human interferon-y in E. coli, Korean Biochem. J . . 19, 266, 1986. 80. Kang, S. M., Kim, S. W., Chung, I. Y., Na, D. S., Klm, J. Y., and Han, M. H., Cloning of human interleuhn-2 cDNA in E. coli

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by using oligonucleotide primers, Korean Biochem. J., 21, 31, 1988. 81. Kim, G. S., Molecular cloning of a cDNA encoding osteocalcin. I. Isolation of osteoblastic bone cells and their expression of osteocalcin, Proc. Mol. Biol. Genet. Korea, 3(1), 113, 1988. 82. Kim, J. S., Park, Y. S., and Yim, J. B., Molecular cloning of a suppressor gene su(s)2 of Drosophila by transposon tagging technique, Korean Biochem. J., 20, 29, 1987. 83. Kim, C. H., Song, S. I., and Choi, Y. D., Regulation of soybean glycinin gene expression: a cDNA cloning, Proc. Mol. Biol. Genet. Korea, 3(1), 143, 1988. 84. Kim, D. L., Park, D. H., and Park, S. K., Cloning and expression of a cDNA encoding a pea chitinase in tobacco, Proc. Mol. Biol. Genet. Korea, 3(2), 129, 1988. 85. Lee, J. S., Choi, Y. H., Sohn, H. W., and Moon, Y. H., Studies on the structure and expression of potato proteinase inhibitor I1 genes, Proc. Mol. Biol. Genet. Korea, 3, 131, 1988. 86. Cho, M. J., Yun, H. D., Choi, Y. L., Choi, Y. J., Lee, K. S., and Brill, W. J., Molecular cloning of alfalfa (Medicago sariwa L. vernal) leghemoglobin structural genes3ynthesizedby poly(A)+ -mRNA from alfalfa root nodules, Korean Biochem. J., 18, 77, 1985. 87. Ki, W. K., Song, J. Y., Cho, T. Y., Shim, K. H., and Cho, S. H., Base sequence of autonomous replicating segment of Candida tropicatis, Proc. Mol. Biol. Genet. Korea, 3(1), 21, 1988. 88. Park, M. Y., Biochemistry of cellulase genes, Proc. Ann. Meeting Korean Biochem. Soc., 1987. 89. Lee, H. S., Yoon, S. C., Lee, C. W., and Lee, S. Y., DNA sequencing characterization of MAK 18 gene of Saccharomyces cerevisiae, Proc. Mol. Biol. Genet. Korea, 2, 47, 1987. 90.Kim, Y. S., Hyun, S. W., and Rho, H. M., Nucleotide sequence of the core antigen-coding region of hepatitis B virus, Korean Biochem. J., 18, 304, 1985. 91. Kim, K. T., Hyun, S. W., Kim, Y. S., and Rho, H. M., Complete nucleotide sequence of hepatitis B virus (subtype. adr), Korean Biochem. J . , 21, 319, 1988. 92. Chung, D. H., Nishiwaki, K., and Oshirna, Y., Studies on the his5 gene of yeast. The nucleotide sequence of 5’ upstream region of the his5 gene of Saccharomyces cerevisiae, Korean J . Appl. Microbiol. Bioeng.. 13, 19, 1985. 93. Hong, H. J., Choi, E. H., YOO,S . H., and Yoo, 0. J., Isolation and sequence analysis of a gene encoding human serine tRNA, Korean Biochem. J.. 20, 143, 1987. 94. Kim, Y. J., Lee, B. J., and Kang, H. S., The studies on the organization and expression of tRNA gene. 11. The structure of the tRNAasp of Aspergillus nidulans, Korean Biochem. J . , 19, 112, 1986. 95. Koh, M. J., Park, I. W., and Lee, S. Y., Sequence of 5s rRNA of Pseudomonas putida and its possible secondary structure, Korean Biochem. J . , 19, 61, 1986. 96. Johng, T. N., Joung, Y. H., and Park, I. W., The nucleotide sequence and the secondary structure of 5 s rRNA from a green algae Chaetomorpha moniligera, Korean Biochem. J . , 20, 83, 1987. 97. Lee, Y. H., Jeon, E. S., and Park, C. U., DNA sequence of the Drosophila melanogaster hsp70 genes affecting heat shock response, Korean Biochem. J . , 21, 339, 1988. 98. Kim, T. K., Choi, C. Y., and Rho, H. M., Construction of the stable and high copy number yeast vectors, Korean J. Appl. Microbiol. Bioeng., 16, 476, 1988. 99. Hwang, D. J., Rhe, S. K., and Park, M. Y., Construction of plasmids vectors, Zymomonas mobilis. Korean J . Appl. Microbiol. Bioeng.. 15, 319, 1987. 100.

Lee, Y. E., Lee, B. J., and Kang, H. S., Characterizationof plasmids

of Zymomonas mobilis and construction of E . coli-Zymomonas shuttle vector, Korean J . Microbiol., 23, 56, 1985. 101. Lee, D. K. and Rho, H. M., Construction of expression vectors

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containing portable P,-promoter of A-phage, Korean Eiochem. J., 17, 177. 1984. 102. Kwon, D. H., Suk, J. S., and Byeon, W. H., Cloning of ori region of R-plasmid pSBK203 and construction of new shuttle vectors for E. coli and E . subtilis using cloned fragments, Korean J. Microbiol., 25, 262. 1987. 103. Cho, M. J., Yang, M. S., Yun, H. D., Choe, Y. L., Choi, Y. J., and Shin, P. G., Construction of broad host range vector for Rhizobium, Proc. Mol. Biol. Genet. Korea, 1, 193, 1986. 104. Park, W., Lee, J. M., Lee, J. Y., Sang, B. H., andHong, S. D., Construction of conditional expression vector by using lambda phage, Proc. Mol. Eiol. Genet. Korea, 1, 179, 1986. 105. Ki, W. K., Yun, S. J., Seok, K. Y., Lee,Y. C., and Cho, Y. U., Studies on yeast vector system for eukaryotic gene cloning, Proc. Mol. Eiol. Genet. Korea, I , 87, 1986. 106. Kim, Y. S., Song, 0. K., and Rho, H. M., Construction of the vector for the cloning of bluntended DNA fragments, Korean J. Genet., 6. 63, 1984. 107. Kim, S. H., Seo,T. S., Kho, Y. H., and Rho, H. M., Development of cloning vector in Corynebacterium glutamicum using natural plasmid, Korean J. Appl. Microbiol. Bioeng., 16, 73, 1988. 108. Choi, Y. C., Lee, S. C., Han, M. H., and Yu, M. H., Optimization of the P-galactosidase polypeptide length for the overproduction of fusion proteins in E. coli, Korean Biochem. J . , 21, 102, 1988. 109. Chung, M. H., Kim, S. H., Jun, H. S., and Rho, H. M., Construction of an expression vector with SV40 DNA in a mammalian cell, Korean J . Microbiol., 25, 165, 1986. 110. Ki, W.K.,Yun,S.J.,Seok,K.Y.,Lee,Y.C.,andSong,J.Y., Studies on yeast vector system for eukaryotic gene cloning. Improvement of chromosomal autonomous replicating vector of yeast, YRp7, Korean J. Appl. Microbiol. Bioeng., 15, 217, 1987. 111. Ki, W. K., Seok, K. Y., Yun, S. J., and Song, J. Y., Studies on yeast vector system for eukaryotic gene cloning. New cloning vector containing autonomous replicating segment from Candida tropicalis, Korean J . Appl. Microbiol. Bioeng., 15, 224, 1987. 112. Jong, K. B., Kim, K. T., Kim, J. Y., Rhee, S. K., and Han, M. H., Expression of hepatitis B virus surface antigen gene in yeast using GALI-ADCI, GACIO, and ADCI promoter, Korean Eiochem. J., 20, 163, 1987. 113. Kim, C. S. and Kim, M. H., Nucleotide sequence determination and expression of X open reading frame of a hepatitis B virus adr subtype, Korean Eiochem. J., 21, 332, 1988. 114. Yun, H. Y., Han, M. H., and Hahm, K. S., Immunoaffinity purification and receptor assay for polymerized human serum albumin of pre-S2 peptide on hepatitis B virus, Korean Biochem. J . , 21, 412, 1988. 115. Korean Genetic Engineering Research Association, Genet. Eng. Q., 24, 101, 1988. 116. Kang, S. M., Kim, S. W., Ha, H. J., Na,D. S.,Park, S. H., Kim, J. Y., and Han, M. H., Overproduction of human interleukin-2 in E. coli, Korean Eiochem. J . , 21, 38, 1988. 117. Yun,H. Y.,Choi,H.L.,Lee,M. K.,Kim,S.H.,Na,D.S.,Lee, S. B., Han, M. H., and Hahm, K. S., Recombinant human interleukin-2. I. Purification and biochemical characterization, Korean Eiochem. J . . Vol. 21. 120. 118. Choi, H. L., Yun, H. Y., Talmadge, J. E., and Hahm, K. S., Recombinant human interleulsin-2. 11. Biological and therapeutic activities, Korean Biochem. J., 21, 127, 1988. 119. Park, H. S., Jung, M. Y., Oh, M. S., Koh, J. H., Kim, H. S., and Hyum, H. H., Isolation and purification of human interferon y from recombinant E. coli monoclonal antibody column and HPLC gel filtration, Korean Eiochem. J., 20, 336, 1987. 120. Park, B. C., Lim, H. S., Kim, M. H., and Lee, D. S., Study on human insulin. I. Synthesis and cloning of genes of human insulin A and B chains, Korean Eiochem. J . , 21, 214, 1988.

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210. Cho, M. J., Yang, M. S., Yun, H. D., Choe, Z. R., Choe, Y. L., and Kang, K. Y., Selection of Rhizobium japonicum mutants having greater symbiotic nitrogen fixing activity with soybean, Korean I . Appf. Microbiol. Bioeng., 13, 79, 1985. 21 1. Chang, W. H., Lee, W. K., Kim, S. Y., and Park, J. B., Production of the monoclonal antibodies to the Escherichia coli heat-stable enterotoxin, J. Korean SOC.Microbiol., 22, 377, 1987. 212. Cho, M. J., Chang, W. H., Choi, M. S., Kim, I. S., Kang, J. S., Park, K. H., Kim, H. K., Cha, C. Y., Chung, H. K., and Rhee, K. H., Purification of heat-labile enterotoxin from an enterotoxigenic Escherichia coli of human origin by monoclonal immunoaffinity chromatography, J. Korean Med. Sci., 2, 65, 1987. 213. Kim, M. K., Cho, M. J., Park, K. H., Lee, W. K., Kim, Y. W., Choi, M. S., Park, J. S., Chang, W. H., and Chung, H. K., Development of assay methods for enterotoxin of Escherichia coli employing the hybridoma technology, J. Korean SOC.Microbiol., 21, 151, 1986. 214. Kim, U. H., Kwon, I. K., An, S. H., and Yoon, Y. D., Studies on microencapsulation of hybridoma cells, monoclonal antibody production against pili antigens of enterotoxigenic E. coli(ETEC) and diagnostic uses, Proc. Mol. Biol. Genet. Korea, 2, 199, 1987. 215. Kim, Y. J., Cho, M. J., Lee, W. K., Park, C. G., Kook, Y. H., and Cha, C. Y., Production and characterization of monoclonal antibodies against Salmonella typhimurium 0-antigen, J . Korean SOC. Microbiol., 23, 319, 1988. 216. Kim, H. D., Kang, S. S., and Ju, J. W., Immune response of BALB/ c and production of monoclonal antibodies against outer membrane proteins from Vibrio vulnificus. J. Korean SOC. Microbiol., 23, 365, 1988. 217. Chang, W. H.,Park, K. H., and Lee, J. B., Serological analysis of Leptospira interrogans isolated in Korea by monoclonal antibodies, J. Korean SOC.Microbiol., 23, 277, 1988. 218. Park, K. H. and Chang, W. H., Serovar identification of Leptospiral strain HS-7 isolated in Korea by monoclonal antibodies, J. Korean SOC. Microbiol., 23, 293, 1988. 219. Choi, C. Y., Yim, G . B., Kim, J. H., Lee, C. G., and Ahn, I. S., Monoclonal antibody production by hybridoma in two forms of bioreactor, Proc. Mol. Biol. Genet. Korea, 2, 225, 1987. 220. Choi, T. Y., Kim, T. Y., Kim, C. W., Kim, K. H., Hwang, E. S., Cha, C. Y., and Kim, K. H., Production of monoclonal antibody to Chlamydia rrachomatis, J. Korean, SOC. Microbiol., 22, 197, 1987. 221. Kim, Y. W., Cho, M. K., Min, C. H., and Yoon, C. S., Isolation and characterization of Rickertsia typhi from patients in Korea, J . Korean, SOC. Microbiol., 23, 265, 1988. 222. Choi, T. K., Paik, T. H., Park, J. K., and Kim, H. J., Studies on the immunochemical characterization of mycobacterial antigens by monoclonal antibodies to Mycobacterium tuberculosis antigen, J . Korean, SOC. Microbiol., 23, 473, 1988. 223. Rhee, K. H., Cho, M. J., and Kim, J. B., Development of monoclonal antibody for diagnosis of tuberculosis, Proc. Mol. Biol. Genet. Korea, 2, 211, 1987. 224. Cha, C. Y., Hwang, E. S., and Kook, Y. H., Production and characterization of monoclonal antibodies specific to herpes simplex virus, J . Korean SOC. Microbiol., 23, 505, 1988. 225. An, S. H. and Kim, U. H., Monoclonal antibodies against Marek’s disease virus and herpes virus of turkeys, J . Korean SOC. Microbiol.. 13, 55, 1983. 226. Cha, C. Y. and Hwang, E. S., Production and characterization of monoclonal antibodies reactive with human cytomegalovirus, J. Korean SOC.Microbiol., 23, 495, 1988. 227. Chung, H. G . and Kim, S. R., Production of monoclonal antibody to hepatitis B surface antigen and its application to radioimmunoassay, Proc. Mol. Biol. Genet. Korea. 2, 165, 1987.

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Critical Reviews In 228. Chung, H. K., Shin, H. S., Kim, C. Y., and Lee, C. H., Change xn pre-S2 epitope of hepatitis B surface antigen during the course of disease as monitored by radioimmunometric assays employing monoclonal antibodies, Proc. Mol. Biol. Gener. Korea, 3(2), 75, 1988. 229. Kim, G. R., Kim, T. G., Rhyu, M. G., b d Lim, B. U., Study on the pathogenesis of hataan virus with monoclonal antibodies, J. Korean, Sac. Microbiol., 22, 1, 1987. 230. Choi, C. O., An, S. W., Lee, C. G., and Cho, S. M., Production of monoclonal antibody to infectious laryngotracheitis virus by cell fusion, Proc. Mol. Eiol. Genet. Korea, I, 251, 1986. 231. Choi, C. O., Lee, C. G., An, S. H., and Keon, J. H., Production of monoclonal antibody to avian infectious bronchitis virus, Proc. Mol. Biol. Genet. Korea, 3(2), 8 3 , 1988. 232. Choi, C. O., An, S. W., Lee, C. G., Cho, S. M., and Na, J. S., Production of monoclonal antibody to newcastle disease virus and its diagnostic use, Proc. Mol. Biol. Genet. Korea, 2, 219, 1987. 233. Kwon, I. K., Kim, U. H., and Park, S. B., Studies on microencapsulation and immobilization of anti-TGEV hybridoma cells and monoclonal antibody production, Proc. Mol. Biol. Genet. Korea, 3, 47, 1988. 234. Kim, U. H., Kwon, I. K., Lee, J. H., and Cho, D. H., Monoclonal antibody production against bovine viral diarrhea virus and development of diagnostic technique, Proc. Mol. Eiol. Gene?. Korea, I , 229, 1986. 235. Cho, S. Y., Kang, S. Y., and Kong, Y., Preparation of sensitive and specific antigen for serodiagnosis of cysticercosis by gel filtration and affinity chromatograph using monoclonal antibody, Proc. Mol. Eiol. Gener. Korea, 3(2), 101, 1988. 236. Kang, S. S., Park, T. K., Cho, D. T., Song, B. H., and Bang, 0. S., Production and characterization of monoclonal antibodies against tumor-specific antigen, alpha-fetoprotein, Proc. Mol. Biol. Genet. Koreo, 2, 183, 1987. 237. Kang, S. S., Park, T. K., Song, B. H., and Cho, D. T., Production of monoclonal antibodies against tumor specific antigen, alpha-fetoprotein. Proc. Mol. Eiol. Genet. Korea, I , 243, 1986. 238. Lee, J. H., Chung, H. K., and Kim, S. W., Purification of carcinoembryonic antigen from culture supernatant of human colon cancer cell line, LS 174T using monoclonal immunoaffinity chromatography, Korean J. Eiochem.. 20, 23, 1988. 239. Lee, J. H., Lee, S. Y., and Cho, D. H., Human-human hybridoma secreting monoclonal antibodies specific to human carcinoma, Proc. Mol. Biol. Gener. Korea, 2. 173, 1987. 240. Cha, C. Y., Hwang, E. S., Cho, M. J., Park, M. H., Ahn, H. S., Kim, N. K., Chang, W. H., and Lee, M. H., Production and characterization of lymphocytic hybridomas secreting monoclonal antibodies reactive with the cell surface antigens of acute lymphoblastic leukemia, J. Korean Med. Assoc., 27, 253, 1984. 241. Kim, H. D. and Kim, K. W., Immunological study on the surface antigens of tumor cells, Proc. Mol. Biol. Genet. Korea, 3(2), 107, 1988. 242. Chung, G. S., Kim, S. T., Hwang, E. S., Chang, W. H., Cha, C. Y., Lee, M. H., and Rhee, K. H., Production and characterization of monoclonal antibodies reactive to human T lymphocytes, J . Korean Soc. Microbiol., 20, 237, 1985. 243. Lim, D. G., Hwang, E. S., Kook, Y. H., and Cha, C. Y., Production and characterization of monoclonal antibodies specific to human Tlymphocytes, Proc. Mol. Biol. Genet. Korea, 2, 191, 1987. 244. Lim, D. G., Hwang, E. S., Park, C. G., Kook, Y. H., and Cha, C. Y., F'roduction and characterization of monoclonal antibodies reactive to human T-lymphocyte subsats, J. Korean SOC.Microbiol., 23, 407. 1988. 245. Kim, S. Y., Chung, H. K., and Tchai, B. S., Enzyme immunoassay for total estrogen using a monoclonal antibody, Korean J. Biochem., 20, 33, 1988.

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246. Suh, P. G., Chung, H. K., and Tchai, B. S., Production and characterization of monoclonal antibodies to estrogen, Korean J . Biochem., 17, 191, 1958. 247. Whang, T. E. and Lim, H. O., Production of monoclonal antibodies against abscisic acid and its application, Proc. Mol. Biol. Gener. Korea, 3(2), 95, 1988. 248. National Bureau of Statistics, Economic Planning Board, Republic of Korea: Annual Report of the Cause of Death Statistics (based on vital registration), 1986. 249. Yu, G. and Kim, C. Y., Epidemiological consecutive studies on incidence and status of HBsAg carriers among university employees in Korea, Korean J. Inr. Med., 23, 649, 1980. 250. Rhee, K. H., Cho, M. J., and Kim, J. B., Ministry of Health and Social Affairs, Korean National Tuberculosis Association: The 5th Tuberculosis Prevalence Survey in Korea, 1985. 43. Production and purification of recombinant antigens of Mycobacterium tuberculosis, Proc. Mol. Eiol. Genet. Korea, 3(2), 69, 1988. 25 1. Rhee, K. H., Cho, M. J., and Kim, J. B., Production and purification of recombinant antigens of Mycobacterium ruberculosis, Proc. Mol. Biol. Gener. Korea, 3(2), 69, 1988. 252. Chang, W. H., Kim, S. Y., and Seo,J. S., Restriction endonuclease DNA analysis of leptospiral field isolate from Korea, J. Korean SOC. Microbiol., 22, 463, 1987. 253. Goh, G. D., Chung, K. S., and Lee, K. M., Studies on egg transfer in Korean native cattle. Ill. Non-surgical egg transfer following superovulation and egg transfer, Korean J. Anim. Sci., 23, 33 I , 1981 . 254. Gu, J. H. and Chung, C. K., Experiments on non-surgical embryo recovery and transfer in dairy cow, J. Korean Ver. Med. Assoc., 10, 45, 1982. 255. Seok, H. B., Lee, K. W., Shin, Y. S., Kim, H. J., Cho, Y. Y., Oh, D. G., Chee, S. H., Im, K. S., Im, R. D., and Baker, R. D., lnfluences of frozen embryos on conception in cattle. 11. Effects of two-step equilibrium in sucrose suspending medium, Korean J. Anim. Sci., 25, 430, 1983. 256. Chung, K. S., Yoon, J. S., Lee, H. T., Yoo, S. H., and Kim, C. I., Twin induction by embryo transfer in cattle. V1. Calving rate following non-surgical transfer of fresh and frozen embryos, Korean J. Anim. Sci., 25, 424, 1983. 257. Kim, H. S., Kim, Y. J., Lee, J. M., Lee, K. S., and Chung, K. S., Studies on the factors affecting superovulation induction in cattle, Korean J. Anim. Sci.. 27, 201, 1985. 258. Kim, H. S., Oh, S. J., Yang, B. S., Yoo, S. H., Kim, J. C., Baig, S. Y., and Lee, K. S., Studies on induction of superovulation and embryo transfer in cattle, Korean J. Emb. Trans., 1, 69, 1986. 259. Lee, J. H., Suh, T. K., and Park, H. K., Factors affecting superovulatory responses in dairy cows, Korean J. Emb. Trans.. 2, 27, 1987. 260. Im, K. S., Kim, C. K., Voss, H. J., Allen, S., Zheng, X., and Foote, R. H., Sexual behaviors, estrus detection and conception of heifers synchronized by progesterone intravaginal device (PRID) and synchromate-B, Korean J. Anim. Reprod., 9, 140, 1985. 261. Oh, S. J., Yang, B. S., Kim, H. S., Lee, K. S., Kim, K. S., Spears, J., and Oury, H. M., A study on the synchronization of estrus and the frozen embryo transfer in cattle, Korean J. Anim. Sci., 28, 468, 1986. 262. Lee, J. H., Park, H. K., and Cho, M. H., Interactions between recipients and embryos affecting pregnancy rates in bovine embryo transfer, Korean J . Emb. Trans., I , 76, 1986. 263. Kim, H. S., Oh, S. J., Yang, B. S., Lee, K. S., and Chung, K. S., Studies on the non-surgical embryo recovery and transfer in cattle, Korean J. Anim. Sci., 27, 206, 1985. 264. Kim, H. S., Oh, S. J., Yang, B. S., Yoo,S. H., Kim, J. G., and Lee, K. S., A study on the factors affecting the survival of embryos transferred in cattle, Korean J . Anim. Sci., 28, 578, 1986.

Volume 10. Issue 1


Biotechnology 265. Lee, J. Y., Chung, K. S., and Kim, J. B., Separation of X- and Ybearing spermatozoa. IV. Separation of bull spermatozoa by the combination of density gradient centrifugation and Sephadex gel filtration, Korean J. Anim. Reprod., 12, 141, 1988. 266. Song, D. J. and Park, C. S., A study on induction and synchronization of estrus in Korean native goats, Korean J . Anim. Sci.,26, 13, 1984. 267. Park, C. S., Choe, S. Y., Lee, H. J., and Lee, J. S., Studies on the technological development of embryo transfer and manipulation in goats. I. Estrous induction, short estrous cycle and superovulation in goats, Proc. Mol. Biol. Genet. Korea, I , 215, 1985. 268. Park, C. S., Choe, S. Y., Lee, H. J., Lee, J. S., and Park, H. S., Studies on the technological development of embryo transfer and manipulation in goats. 11. Production of monozygotic twins by bisection of mouse and goat embryos, Proc. Mol. Biol. Genet. Korea, 2, 157, 1987. 269. Na, J. S., Kim, Y. S., and Kang, B. K., Embryo transfer in Korean native goat, Korean, J . Anim. Sci., 29, 295, 1987. 270. Park, C. S., Choe, S. Y., Lee,H. J., Lee, J. S., and Park, H. S., Studies on the technological development of embryo transfer and manipulation in goats. 111. Improvement of viability and conception rate following bisection and transfer of mouse and goat embryos, Proc. Mol. Biol. Genet. Korea, 3 , 9, 1988. 271. Kim, N. H., Chung, K. S., Rho, H. C., Pek, U. H., and Lee, K. K., Production of monozygotic twin mice by bisecting morula, Korean J . Anim. Sci., 28, 527, 1986. 272. Hwang, W. S., Studies on the production of twins in cattle, Proc. Mol. Biol. Genet. Korea, 3(2), 27, 1988. 273. Yoon, M. S. and Chung, K. S., The freezing of mouse embryos, Korean J . Anim. Reprod., 8, 116, 1984. 274. You, J. H. and Lee, J. K., The study on the freezing methods and the cryoprotectants for rat embryo preservation, Korean J . Anim. Reprod., 8, 22, 1984. 275. Jin, D. I., Im, K. S., Ohh, B. K., and Lee, Y. B., Effect of cryoprotectants, ice induction, cooling rate and storage length on the survival of mouse embryos, Korean J . Anim. Sci., 28, 474, 1986. 276. Jo, C. H., Cheong, C. K., and Hwang, W. S., Studies on the effects of ethylene glycol on the survival of frozen-thawed mouse embryos, Korean J . Vet. Res., 27, 331, 1987. 277. Rho, H. C., Chung, K. E., Shin, G. Y., Chung, B. H., Pek, U. H., and Chung, K. S., Studies on the industrial utilization of frozen bovine embryos, Korean J. Anim. Sci., 30, 151, 1988. 278. Smk, H. B., Lee,K. W., Oh, S. Y., Son, D. S., Yun, C. K., Yun, H. J., Kim,H. J.,Cho, Y. Y.,Oh,D. K.,Che,S. H.,Im, K.S., and Mahon, G. D., Influences of frozen embryos conception in cattle. 111. Effects of surgical transfer of ova rehydrated by five-steps for glycerol elimination, Korean 1. Anim. Sci., 26, 423, 1984. 279. Kim, J. K., Kim, C. K., Kang, M. J., Kim, Y. H., and Kang, M. S., Studies of simplified procedures for freezing and thawing of bovine embryos. 111. Effects of simplified procedures of freezing and thawing using a glycerol diluent containing sucrose on the rabbit embryos survival rate, Korean J. Anim. Sci.,30, 576, 1988. 280. Kim, J. K., Kim, C. K., Kang, M. J., Chang, D. J., and Kim, S. H., Studies on simplified procedures for freezing and thawing of bovine embryos. IV. Effects of simplified procedures of freezing and seeding using a cryoprotectant containing sucrose on the rabbit embryo survival rate determined with the FDA test, Korean J . Anim. Sci., 30, 583, 1988. 281. Lee, J. H., Effect of embryo and recipient condition of pregnancy rate following bovine embryo transfer in southern Korea, Theriogenology, 29, 268, 1988. 282. Shim, H. S., Kim, J. H., Lee, B. C., Kim, J. B., Park, H. Y., and Chung, K. S., Studies on the production and applications of monoclonal antibodies to H-Y.1. Production of monoclonal antibodies to H-Y,Korean J . Anim. Sci., 30, 411, 1988.

1990

283. KO, J. J., Shim, H. S., Kim, J. B., Park, H. Y., Chung, K. S., and Lee, K. K., Effect of H-Y antibody on in vitro development of mouse embryos, Korean J . Anim. Reprod., 10, 42, 1986. 284. Han, Y. M., Kim, J. B., Park, H. Y., Chung, K. S., and Lee, K. K., Sexing of mouse embryos by chromosomal analysis, Korean J. Anim. Reprod., 10, 36, 1986. 285. Yang, B. K. and Kim, C. I., Study on the sexing of preimplantation mouse embryo exposed to H-Yantisera. I. Sexing of mouse embryos by cytolytic assay, Korean J. Anim. Reprod., 12, 31, 1988. 286. Kim, S. K., Studies on the control of sex ratio by treatment of protein column and H-Yantibody in ewes, Korean J . Anim. Reprod., 12, 42, 1988. 287. Yang, B. K., Chang, C. S., and Kim, C. I., Study on the sexing of preimplantation mouse embryo exposed to H-Yantisera. 11. Sexing of mouse embryos by immunofluorescence assay, Korean J. Anim. Reprod.. 12, 37, 1988. 288. Goh, G. D., Hong, B. J., Yang, B. K., and Kim, C. I., Immunofluorescent detection of H-Y antigen on preimplantation bovine embryos, Proc. Mol. Biol. Genet. Korea, 3(2), 5 , 1988. 289. Chung, H. M.,Park, Y. S., Chung, K. S., and Lee, K. K., Studies on nuclear transplantation in mouse embryos. I. In vitro development of nuclear transplanted mouse embryos, Korean J . Anim. Sci., 30, 452, 1988. 290. Lee, C. S., Park, H. D., Chung, K. S., and Lee, K. K., Production of nuclear transplanted mice, Korean J. Anim. Sci., 31, in press, 1989. 291. Lee, K. K.,Han, Y. M.,Uk, N. G.,Lee, C. S.,Kim,Y. J.,Kim, J. M., Kim, J. Y., and Han, M. H., Expression of human growth hormone gene in transgenic mice, Korean J . Anim. Sci.. 31, in press, 1989. 292. Lim, H. S., Park, B. C., Kim, M. H., and Lee, D. S., Chemical synthesis, and cloning of human proinsulin gene, in Besr Approach to the [deal Therapy of Diabetes Mellitus, Shigeta Y.,Lebovitz, H. E., Gerich, J. E., and Mdaisse, W. J., Eds., Elsevier, New York,1987, 245. 293. Han, H. G. and Yang, C. H., Purification and chemical modification of a-amylase from Bacillus amyloliquefaciens, Proc. Mol. Biol. Genet. Korea, 2, 267, 1987.

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Genetic engineering of microorganisms for biotechnology.

Engineering synergy in biotechnology.

Frontiers in biomedical engineering and biotechnology.

Physical medicine and rehabilitation in South Korea: past, present, and future.

[Directions of an ethic in genetic engineering: evaluation of an ethic for biotechnology].

[Korea]ICT and Healthcare in Korea: Present and Prospect.

Critical Care In Korea: Present and Future.

Evaluation of riboflavin intakes and status of 20-64-year-old adults in South Korea.

Current status of infrastructures of obstetrics and gynecology in South Korea.

South Korea.

Status, alert system, and prediction of cyanobacterial bloom in South Korea.

Effects of Health Status and Health Behaviors on Depression Among Married Female Immigrants in South Korea.

Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis.

Current status of and strategies for hepatitis C control in South Korea.

An emerging interface between life science and nanotechnology: present status and prospects of reproductive healthcare aided by nano-biotechnology.

Engineering considerations for the application of extremophiles in biotechnology.

Current Status of Management in Type 2 Diabetes Mellitus at General Hospitals in South Korea.

Genetic diversity and population structure of the long-tailed goral, Naemorhedus caudatus, in South Korea.

Corrigendum: Mitochondrial genetic diversity, phylogeny and population structure of Hydropotes inermis in South Korea.

Mitochondrial genetic diversity, phylogeny and population structure of Hydropotes inermis in South Korea.

Green Chemistry at the present in Korea.

Current status of robotic thyroid surgery in South Korea: a web-based survey.

Current Status of Atopic Dermatitis-Related Information Available on the Internet in South Korea.

A survey on the status of hepatitis e virus infection among slaughterhouse workers in South Korea.