university of maryland task force on high technology

University

of

Maryland

Task Force

High Technology/

Biotechnology

842853

University

of

Maryland

Task Force

on

High Technology/

Biotechnology

Report

This report by the University of Maryland Task Force on High Technology/Biotechnology was submitted to the Educational Policy Committee of the Board of Regents at their meeting on November 18,1983. Address inquiries to Dr. Rita R. Colwell, Vice-President for Academic Affairs, Central Administration, University of Maryland, 3300 Metzerott Road, Adelphi, Maryland 20783.

Photography by Skip Brown Design by Erica Brown

Dr. Shain Down-Kung and Dr. Paul Lovett (UMBC) are examining

an autoradiogram showing the DNA sequence of a plant

chromosome.

Executive Summary v

Task Force on Biotechnology vi

Introduction 1

The Maryland Biotechnology Institute: The University/Industry Relationship 3

University Programs and Facilities Required for Graduate Training and Research in Biotechnology 6

Basic and Applied Molecular Biology 6 Biochemical Engineering Laboratory 8 Plant Biotechnology 9 Biotechnology and Human Vaccine

Development 10

Biotechnology Applications in Agriculture

and Fisheries 12

Bioelectronics, Biosensors, and Artificial

Intelligence 13

Marine Biotechnology 15

Biophysical Technology—A Cooperative

Program 20

Summary 23

iii

Executive Summaiy

The Task Force on High Technology and Bio- technology recommends establishing a center of excellence in biotechnology—the

Maryland Biotechnology Institute—as a separate unit of the University of Maryland. Biotechnology and its rapidly expanding application to high technology industries promises to have enormous economic benefits to those areas of the country that have universities with the expertise to solve diverse high technology problems, and to seek new opportunities. The Maryland Biotechnology Insti- tute will consolidate the expertise now at the Uni- versity; it will work in partnership with high technology industry now in Maryland and serve as a magnet for attracting new industry, thus providing the State of Maryland with the scientific component to its commitment to high technology industries. The consequent economic benefits—increasing employment and broadening the tax base—will depend on the strength of biotechnology capabilities at the University of Maryland. This proposal by the Task Force provides evidence that the University is in a favorable position to become a national and international leader in biotechnology.

Throughout the University of Maryland campuses—in College Park, Baltimore and Catons- ville—Maryland researchers are engaged in biotechnology projects that have already achieved national recognition. But that work is dispersed in different departments and laboratories. Although research is funded for particular projects, too often little attention can be given to the potential of broad-based interdisciplinary programs for solving complex problems that new high technology industries require. The center of excellence in biotechnology at the University of Maryland will make possible more cohesive approaches to solving those problems. While academic programs are administered through the University, the Mary- land Biotechnology Institute will provide adminis- trative coordination within the University, will serve to catalyze the development of biotechnology initiatives in response to state, federal and

public needs, and will work with industries to apply broad biotechnology expertise to pressing issues of concern to the region and nation.

To consolidate biotechnology projects administratively under the Institute will require a commitment by the state, as a first phase for attracting industrial sponsorship, to support expansion of academic and research programs in biotechnology. This Task Force report summarizes the need for a biotechnology center; the benefits of partnership with industry to the University and state; and the specific University programs and facilities that require expansion in basic and applied molecular biology, biochemical engineering, plant biotechnology, human vaccine development, agriculture and fisheries, artificial intelligence, marine studies and biophysical technology.

V

Task Force on

Dr. Rita R. Colwell, Chairman Vice President for Academic Affairs University of Maryland Central Administration

Dr. Filmore E. Bender Associate Director and Professor Agricultural Experiment Station Room 1122, Symons Hall University of Maryland College Park Campus

Dr. George E. Dieter Dean, College of Engineering Room 0102, Surge Facility University of Maryland College Park Campus

Dr. Edward V. Ellis Vice Chancellor for Academic Affairs University of Maryland Eastern Shore Campus Princess Anne, Maryland 21853

Dr. Stephen E. Forrer Dean, University College Room 3124B, Center of Adult

Education College Park Campus

Dr. Walter S. Jones Vice Chancellor for Academic Affairs Room 1001, Administration Building University of Maryland Baltimore County Campus

Dr. Sam W. Joseph Chairman, Department of Microbiology Room 2131, Skinner Building University of Maryland College Park Campus

vi

High Technology/Biotechnology

Dr. William E. Kirwan Vice Chancellor for Academic Affairs Room 1119, Main Administration Building University of Maryland College Park Campus

Dr. Shain-Dow Kung Chairman, Biological Sciences Room 433, Biological Sciences University of Maryland Baltimore County Campus

Dr. Myron M. Levine Center for Vaccine Department School of Medicine University of Maryland Baltimore City Campus

Dr. Ian Morris Director, Center for Environmental

and Estuarine Studies University of Maryland, Box 775 Cambridge, Maryland 21613

Dr. David A. Nagey Obstetrics and Gynecology School of Medicine Room N6E11, North Hospital Building University of Maryland Baltimore City Campus

Dr. Cyril A. Ponnamperuma Department of Chemistry Room 3130, Chemistry Building University of Maryland College Park Campus

Mr. Robert G. Smith Vice President for University Relations University of Maryland Central Administration

Dr. David S. Sparks Vice President for Graduate Studies

and Research University of Maryland Central Administration

Dr. Larry N. Vanderhoef Provost, Divison of Agriculture

and Life Sciences Room 1104, Symons Hall University of Maryland College Park Campus

Dr. Mary W. Burger, Staff Assistant Vice President for Academic Affairs University of Maryland Central Administration

Dr. John Hays, Resource Support Department of Chemistry Room 416B, Chemistry and Physics Building University of Maryland Baltimore County Campus

Dr. Leroy Keith, Resource Support Vice President for Policy and Planning University of Maryland Central Administration

Dr. Samuel Price, Resource Support Coordinator of Sponsored Research University of Maryland Central Administration

Dr. Jack Schubert, Resource Support Chairman, Chemistry Chemistry and Physics Building University of Maryland Baltimore County Campus

Dr. Richard E. Wolf, Resource Support Department of Biological Sciences Room 407, Biological Sciences University of Maryland Baltimore County Campus

vii

Introduction

A biotechnology center of excellence at the University of Maryland will create leading edge technology through joint university-

government-industry research in such areas as biochemical engineering, marine biotechnology, plant biotechnology, hybridoma technology, bioelectronics, biophysics and biomathematics. The Maryland Biotechnology Institute will be a significant force in attracting to the State of Maryland outstanding scientists and engineers who will strengthen high technology companies now located in Maryland and participate in the formation of new companies. Moreover, the Institute will enhance the state's attractiveness for firms from other regions of the country, as well as establishment of new companies.

There is strong intellectual breadth in science, engineering and public policy in Maryland. With financial support and participation by industry and by the State of Maryland, the biotechnology center of excellence can lead to significant growth in jobs and in the state's economy. However, if Maryland is to attract its share of the highly sought after technology industry in competition with other aggressive states, it is imperative that we act without delay. Delay will make later competitive entry much more difficult and far more costly. Al- ready, many universities have moved into cooperative agreements with industry in such areas as microelectronics, polymer science, computer graphics, robotics and biotechnology.

In October 1982, the Governor's Ad Hoc Com- mittee on High Technology in Maryland concluded that timely and consistent investment to stimulate high technology industry within the state represents a realistic opportunity for more jobs, a broadened tax base, and the maintenance of a desirable quality of life for Maryland citizens.

A study by the Office of Business and Industrial Development of the Maryland Department of

Community and Economic Development concluded that there are extraordinary opportunities for high technology industry in Maryland—opportunities that could lead to the kind of growth that has had such significant economic benefits for south- ern California. But growth, the study points out, "largely depends on technical innovation, organi- zational development and distribution development." Moreover, "the strength of the University of Maryland will determine the continued vitality of high technology industry in the state."

While Maryland's 200 high tech manufacturing firms and 600 R&D organizations have been successful in attracting well-trained professionals and, in addition to serving state interests, are serving domestic and international markets, the potential for increased growth is well acknowledged. In the last twenty years, high technology industry has ac- counted for 75 percent of the net growth in the country's manufacturing employment, while an- nual employment growth averages as much as 9 percent in some high technology industries. The key to growth, in nearly every case, has been proximity to a major university center.

Strengthening high technology capabilities through development of a biotechnology institute will foster university and industry cooperation and will lead to development of new programs to meet the needs of the state and nation. An effective Uni- versity-industry partnership will create the basis for Maryland's prosperity as we head towards the next century.

The task force of university research faculty at the University of Maryland, working with repre- sentatives of industry and government, proposes formation of a biotechnology institute and con- cludes that an investment in this institute can pro- pel Maryland to a national and international leadership role in biotechnology.

1

A gel is being loaded with radioac- tive DNA to determine its primary sequence.

The Maryland Biotechnology Institute

The University/Industry Relationship

Biotechnology is the engineering application of basic principles of molecular biology to a wide variety of practical problems in such

diverse disciplines as cellular chemistry, pharma- cology, agriculture, aquaculture and biochemistry. To take one example of the application of biotechnology techniques, the microstructure of genes is well understood and production of certain gene copies by the millions have now become a reality. It is possible to start with a protein (the major product of genes) and use methods of biotechnology to produce the gene which made that protein. This work is leading to techniques that will allow insertion of "healthy genes" into gene-defective cells. These techniques are applicable across a range of problems, from improving resistance by plants to disease to providing fish with a "natural" resistance to heavy metals.

Thus, biotechnology is the "cutting edge" of biological/physiological research, including such technologies as monoclonal antibody production, the application of engineering techniques to the solution of biological problems as exemplified by computerized clinical data bases, and the utilization of biological principles in the solution of non-biological problems, such as ongoing attempts to replace non-organic integrated circuits with organic circuits.

Biotechnology—while it relies on the developments of basic research—is an applied science that is already being made use of by a variety of different high technology industries. Needless to say, the many potential benefits have generated much excitement. It is becoming clear, though, that most of the methods of biology must be fine-tuned before they can be used for any large-scale, precise work. Specifically, the fine qualitative work that has been done must be made quantitative by highly trained biophysicists, physical chemists, physi- cists and mathematicians, and put into production line processes by biochemical engineers. It is essential, for example, if biochemical engineers are

to scale-up products currently being made in test tubes, that our biotechnology capabilities be advanced. The State of Maryland has a significant number of biotechnology firms, each of which is facing problems associated with the transition from test tube to production facilities. The Mary- land Institute of Biotechnology will be a service to these firms just as it will serve as a means for information transfer in education—undergraduate, graduate and continuing professional training.

High technology industry needs ever greater multidisciplinary scientific expertise—and the sources of that expertise are the universities which currently receive less than 5 percent of the estimated $5 billion industry spends annually on research. In recent years—with recognition by both universities and industries of the mutual benefits that come from working cooperatively—the funding relationships have begun to change. And Maryland, if it is to be in the forefront of the nation's universities in biotechnology, must take advantage of the changing opportunities for leadership.

The State of Maryland has a

significant number of

biotechnology firms, each

facing problems in the

transition from test tube to

production facilities.

University/Industry research centers have been organized throughout the country, at M.I.T. (Poly- mer Processing Program), the University of Michi- gan (Center for Robotics and Integrated Manufacturing and Molecular Biology Institute), North Carolina State University (Communications and Signal Processing), and Washington Universi-

3

ty (Center for Biotechnology) to name just a few. The advantages to universities are numerous. To begin with, industries provide funds that go toward the support of graduate students and toward the underwriting of laboratory equipment that strengthen the capabilities of university researchers. Furthermore, the practical questions that industry needs to answer pose challenging problems that can help attract the best students to the Uni- versity who will then gain the expertise that can lead to research and corporate positions after graduation. Moreover, the increased funding that comes with study of applied problems is a stimu- lus to basic research, which increases the capability and scientific strength of university faculty researchers. This, in turn, makes the institute a further attraction to new firms, and leads to development of new products, thereby creating new industries for manufacturing and marketing.

Throughout the University of Maryland—and especially at UMCP, UMAB and UMBC—scientists and engineers are at work on diverse biotechnology-related projects in such departments as agron- omy, botany, chemistry, microbiology, dairy science and medicine. Veterinary scientists at UMCP, for instance, are now able to test huge poultry flocks for contagious diseases in one-tenth the time it has taken in the immediate past; this is because the new technology uses very specific antibodies which are able to identify certain diseases that have plagued poultry farmers in Mary- land for decades, just recently, Maryland scientists were able to get an early profile on flocks that had contracted avian influenza. As a result, the Univer- sity of Maryland led the effort (joined now by the state of Pennsylvania and the Maryland Depart- ment of Agriculture) to eradicate this outbreak. In the past without these quick assays, diseases such as this would have been on their way to becoming state-wide epidemics before it was known that the disease was present.

Although ongoing research programs at Mary- land have successfully been developing levels of excellence and have achieved national recognition, individual projects are, in effect, dispersed in different departments and laboratories throughout the university. While separate efforts are producing results, it is clear that greater coordination among the numerous projects would make the

best use of time and talent, especially in interdisciplinary projects. Dispersed projects are often relatively under-funded and common use of certain resources would alleviate this problem. Further, the most efficient means for attracting applied and basic research support from government and industry would be through the Institute of Biotech- nology at the University of Maryland; the Institute will provide the means for coordinating large numbers of ongoing biotechnology research projects while providing a mechanism for bringing together more efficiently the special talent at the university to deal with increasingly complex research problems faced by government and industry.

Throughout the University,

scientists and engineers are at

work on diverse biotechnology-

related projects.

The Maryland Biotechnology Institute will be centrally administered, following the model of the Sea Grant College at the University of Maryland, and will serve the entire state in the management of federal, state and industry funding and in the linkage with other universities, colleges, institutions and private firms of the state. We have demonstrated that the Sea Grant College model offers a cost-effective funding mechanism, as well as efficient project oversight and review. The Institute will be operated by a Director and staff and will be advised by a Board of Directors representing industry, university and government. While academic programs and course offerings are administered through the University, the Office of the Director will cooperate in the development of new academic programs and coursework.

The Office of the Director will serve as a catalyst for stimulating the development of biotechnology initiatives in response to state, federal and public needs. The Director will work with scientific advi- sors and the Board of Directors and legal and business staff to develop the terms of University/ industry sponsorships with regard to funding levels, and exclusive and non-exclusive patent rights

4

to discoveries that result from partnership. The Office of the Director will also serve as the

focus for coordinating support programs with sponsors, cooperating with department and laboratory heads and industry to arrange programs in training. In this connection. University College, a separate campus of the University of Maryland; provides a unique program for the support of biotech education. With the rapidly changing bio-tech industry requiring the frequent transfer of new knowledge as well as the exchange of ideas, the professional Career Development Program at Uni- versity College provides a context, as well as design support, for conferences and seminars which permit such exchanges. The Center of Adult Edu- cation has a unique conference facility designed precisely for this purpose. In addition, the recent creation of the Center for Technology Programs will provide focus for the exchange of ideas, as well as retraining and education. Further, Univer- sity College extends these programs and services with programs at Shady Grove and at other loca- tions throughout the state.

University Programs and Facilities Required

for Graduate Training and Research

in Biotechnology

Basic and Applied Molecular Biology

For the University of Maryland to establish a Biotechnology Institute, it is imperative that unique education and research capabilities

be expanded and existing programs be coordinated administratively. New academic programs to meet the specific needs for biotechnology industries in the area of basic and applied molecular biology need to be developed. While the University will administer the graduate program, inter-campus cooperation at the University of Maryland must be stressed and supported. The concept of a single University-wide Graduate School has proved exceedingly useful. The University-wide graduate programs in Marine Estuarine and Envi- ronmental Sciences (MEES) and Toxicology provide models for graduate training in basic and applied molecular biology and biotechnology. The net result of the arrangements is enhancement of cooperative research activities in pursuit of scarce learning resources and opportunities, and a reduc- tion of duplicative activities. As a result of past experience and because of the powerful motivations for change, the University of Maryland is ready to respond rapidly to the need for a new institute. The basic resources are in place. The University's willingness to move is obvious. The opportunity for taking maximum advantage of an investment in the future economy of the State of Maryland will never be greater.

The objectives of the graduate training in basic and applied molecular biology include expansion of the M.S. program in Applied Molecular Biology established at the University of Maryland, Balti- more County (UMBC), so that more students can be served, a broader range of courses offered, and greater opportunities opened for industry scientists to receive specialized training. A Ph.D. program in both basic and applied molecular biology

will be developed on a University-wide basis, pat- terned after the MEES program, to provide enhanced opportunities for training and research in areas directly relevant to biotechnology, compared with conventional Ph.D. programs. The program will utilize the resources of the entire university and will be centrally coordinated and campus-directed as is the MEES program, a highly successful graduate program now operational throughout the University of Maryland.

Biotechnology companies in the State of Mary- land will participate in the program by placement of industrial interns, by critiques of the training of these interns, and by designation of industry scientists as University of Maryland Biotechnology Fellows who receive training in courses offered in the program. The "full-share" training expenses will be borne by the respective companies. It is expected that the industrial scientists will inform University of Maryland faculty about industry research areas and interest in collaborative and in-

dustry-sponsored research. Scholarship support for students and direct support for specific courses in the program will also be provided by industry.

The rationale for these programs is that they will serve the interests of biotechnology companies in the state, and be incentives to attract new ones, by increasing the number of middle-level professionals with biotechnology skills. The growing pool of biotechnology-oriented Ph.D.-level molecular biologists will increase the University's research capabilities in biotechnology with respect to the variety of specialties and extent of effort. Results will include more research on problems of interest to industry and to the State of Maryland and enhanced funding opportunities from external private capital for basic research. Industry scientists will be able to acquire relevant information and new laboratory skills more efficiently. The highly visible and productive focus of education and research in biotechnology will be a magnet for industrial, federal and institutional support.

The programs will build on a foundation of existing strengths at the University of Maryland. A na- tionally recognized M.S. program in Applied Molecular Biology at UMBC already is underway. The first graduates, judging by their success as summer interns, will have an impact on the Mary- land biotechnology industry from 1984 on. A

strong group of externally funded research faculty in molecular and cellular genetics and related areas are located at UMBC and there is a broad range of microbiological research interest in the Depart- ments of Microbiology and Chemistry at the Uni- versity of Maryland, College Park (UMCP), as well as a variety of educational and research opportunities in the general area of fermentation and biochemical engineering at the University of Maryland, Baltimore City (UMAB) (Department of Pharmacognosy-Microbiology) and UMCP (De- partment of Chemical Engineering). The M.S. program will be expanded to include a larger number of students and a new. University-wide Ph.D. program, involving all the campuses of the University.

A specific aim of a proposed new Ph.D. program, distinguishing it from other programs, is to provide each graduate student in the program, early on, with a broad theoretical knowledge of molecular biology, as well as a breadth of state of the art laboratory skills.

It is frequently the case that Ph.D. students enter the research laboratory before they are pre-

pared to choose a thesis project; consequently, the research problem is chosen by the thesis advisor, often based on and limited by methods employed in the mentor's laboratory. The result is often de- rivative rather than innovative research. The Ph.D. program will, therefore, be designed to enhance the quality of dissertation research by providing students with a theoretical basis for choosing important research problems and the laboratory skills necessary for solving them.

A second aim of the Ph.D. program is to provide graduates with the broad range of knowledge and skills to enable them to be productive research scientists in industry immediately upon graduation. Many biotechnology firms will appreciate that a post-doctoral training period is not always necessary before a scientist can be innovative and productive in the laboratory. Certainly, Ph.D. graduates who desire careers in industry-based research would prefer to be trained well enough so that upon graduation they can begin work.

Students will become proficient and knowledge- able in theoretical aspects of biochemistry, molecular genetics and molecular biology. The laboratory courses will provide the student with technical skills in biochemistry and the state of the art methods of molecular biology.

Biochemical Engineering Laboratory

A Biochemical Engineering Laboratory for the development of full-scale processes using genetically engineered cells and microorganisms will be established with the laboratory's primary function being to assist in transition from the test tube to production facilities for unique bioproducts offered by the new field of high technology.

The major activities of the proposed laboratory will be centered in six areas. These are the development of scale-up technologies for bioprocesses; demonstration of bioprocesses at the bench and pilot plant scale; collection, analysis and correlation of engineering data for bioprocesses; development and evaluation of new bioproduct separation techniques for full-scale processes; completion of technical and economic feasibility studies for proposed bioprocesses; and development of optimal engineering design and control strategies for bioprocesses.

Development of the laboratory will be in three phases. Initially, bench scale experimental facilities will be designed and equipped with instrumenta- tion, computer monitoring and analytical tools essential for establishment of a firm engineering data base for bioprocess design. On-going research at the University of Maryland in growth kinetics, monitoring and control, separation techniques and scale-up, now hampered by the lack of adequate facilities, will be accelerated.

A biological process cannot attain full utility unless it has been taken to the production stage. Raw materials must be brought together with living cells (genetically engineered microorganisms), conditions that favor the biochemical transformation of the raw materials into products must be maintained (in a fermentor), and the product must be isolated and purified.

Biochemical engineering plays

a major role in the development

of production size facilities for

bioproducts.

Subsequently, pilot scale facilities will be con- structed and emphasis will be placed on development of optimal engineering strategies for bioprocess design and demonstration of these strategies at the pilot scale level.

Finally, special isolation facilities will be established to meet FDA standards for production of pharmaceuticals and handling and processing of potential pathogens and genetically modified organisms.

Biochemical engineering plays a major role in development of production size facilities for bioproducts. Fundamental research is required as a part of process development to provide basic understanding of the microbial processes, as well as the engineering data base, necessary for design, operation and control of process equipment. Work in three major areas of research is needed for genetically engineered cells: kinetics, recovery and scale-up. Fundamental understanding of cell ki-

8

netics is required to optimize product formation, recovery and purification; to specify nutrient re- quirements under varying conditions; to design control strategies for cellular processes; and to determine stability of genetically modified enzymes under varying environmental conditions.

Stability of modified cells frequently is critical, as illustrated by plasmid stability during fermentation in large scale production, i.e., ability of trans- formed cells to maintain a recombinant plasmid unchanged during many generations of growth. When recombinant plasmid methods are employed in the fermentation industry, a common problem is maintaining plasmid carriers reliably within the cell population. To optimize stability of recombinant plasmids, biochemical engineers must investigate factors affecting stability of plasmids, including growth rate, genetic characteris- tics of host cell and plasmid, and environment (growth medium) stress imposed on host cells. Ge- netic manipulation in a laboratory is only one factor in the success of a biological industry; biochemical engineering in scaling-up and design- ing production facilities is essential.

The cost of recovering and purifying a product from the fermentation medium can be the most significant of the costs of a fermentation plant. Be- cause of the number of steps involved for purification, compared to those for cell culture, a pressing need exists for careful, systematic study of the design and optimization of recovery for industrial processes.

Scale-up of production facilities for useful chemicals produced by genetically engineered microorganisms and study of design and optimization of recovery processes are only part of the overall goal, which is to achieve optimal engineering design strategies for bioprocesses and to dem- onstrate these strategies at the level of bench and pilot scale production.

The Biotechnology Institute will enable us to achieve these objectives by the modernization of facilities and the pooling of University strength in engineering support.

Plant Biotechnology

The objectives of the plant biotechnology thrust will be the establishment of M.S. and Ph.D. pro-

grams that give the University unique research and development capabilities. There has been little coordinated development in this area at Maryland, a fact that is surprising, given the proximity of plant biologists at UMCP and UMBC to the Belts- ville Agricultural Research Center (BARC), the largest agricultural research center in the USDA. These experts will constitute the plant biotechnology section of the biotechnology center of excellence.

Large population increases and the world's changing political and social climate have resulted in great demands for plant products, for example, nutritious foods, housing materials, clothing, pa- per, medicines. The demand is greater only for energy and environmental conservation—plants are renewable sources of energy. These new demands and challenges can be met with new approaches in plant research. Already we have examples of genetic manipulation and the use of recombinant DNA technology to enhance nutritional quality, productivity and disease resistance in plants. Simi- larly, these new approaches and research tools are elucidating gene structure and function, character- izing photosynthesis, and clarifying the processes of nitrogen fixation. Studies and techniques which were impossible or impractical only a few years ago are now common practice. Many of these new areas are important to our national and international concerns, with agriculture being the most obvious and immediate. We are in a particularly favorable position to build a new industry, to attract funding for investment in these promising areas, and to crystallize our position of leadership.

Genetic manipulation at the whole plant level has led to the development of today's powerful plant breeding techniques. Molecular approaches in plant biotechnology require knowledge of basic biochemical and physiological mechanisms. Plant and cell tissue culture techniques are essential. They are common laboratory practice at UMCP, UMBC and BARC. Using recently developed methods, for example, single foreign genes can be introduced into plant cells, those plant cells used to regenerate whole plants, and the foreign genes passed on to those plants' progeny. Molecular biology methods now being used at UMBC enable identification of specific genes and these will be applied to genetic manipulation of plants at the

gene level. In gene transfer studies, genes are identified, isolated, manipulated and transferred between organisms. Thus, the plant biotechnology program will employ sophisticated molecular, cellular and whole plant techniques, all of which are in use at the University of Maryland and BARC, to coordinate cutting edge research.

The broad-based capabilities of the research faculty at UMCP and UMBC and scientists at the USDA BARC form a strong nucleus of expertise in plant molecular biology, thus providing the foundation for strong relationships with industries such as Genex, Crop Genetics International and Martin Marietta that are developing plant biotechnology programs.

Biotechnology and Human Vaccine Development

The goals of this area of emphasis are to expand molecular genetic approaches for developing vaccines against infectious diseases of major public health importance or of special relevance to the population of the State of Maryland and to utilize the tools of advanced biotechnology for developing rapid diagnostic techniques for infectious diseases.

Genetic engineering allows the construction of weakened or attenuated live vaccines wherein the genetic material for a factor essential to the disease process, e.g., a toxin, can be cloned and characterized and then altered to produce a non-toxic version. The genes dictating this non-toxic version are then reintroduced into the live pathogenic organism to produce a vaccine strain which can replicate in the human host and stimulate protective immunity, but not cause disease. If a living strain is not needed or desirable for a successful vaccine, then the same techniques of genetic engineering can be used to produce large amounts (i.e., on a commer- cial scale) of purified proteins or other substances for use in vaccines. A further refinement in vaccine development involves the use of synthetic peptides. This approach utilizes the techniques of genetic engineering to clone and determine the base sequence of DNA of the genes that encode proteins useful for vaccines. The DNA sequence allows the deduction of the protein's primary structure and computer analysis can determine

what portions of the protein are antigenic and therefore useful for vaccines. Peptides comprising these regions are then synthesized in large amounts for use as synthetic vaccines.

The Center for Vaccine Development (CVD) at the University of Maryland School of Medicine which will become a cornerstone of the new institute is involved in research on several infectious diseases of public health importance throughout

The most current techniques in

biotechnology are being

applied to develop rapid

diagnostic techniques for

diseases of worldwide public

health importance.

the world. Long-term programs exist for the study of cholera, typhoid fever, Escherichia coli diarrhea and influenza. Other projects involve investiga- tions of rotavirus, Rickettsia rickettsii vaccines, Catn- pylobacter jejuni, Shigella vaccines and Vibrio parahaemolyticus.

Among the facilities available in which to pursue these research activities are a 22-bed isolation ward within the University of Maryland Hospital and a series of specialized laboratories for tissue culture, enteric bacteriology, immunology, electron microscopy, bacterial genetics, rapid diagnostic techniques and pathogenesis. There is also an out- patient facility in Baltimore; a satellite out-patient vaccine testing facility in the Student Health Cen- ter at the College Park campus of the University of Marland; a field area in Santiago, Chile, for testing vaccines against typhoid fever and for studying the epidemiology of endemic S. typhi infection; a field area in Lima, Peru, for epidemiologic studies of infant diarrhea; and a computerized data processing unit.

Some of the major basic science activities include the following; development by recombinant DNA techniques of a patented process for attenuating Vibrio cholerae—large-scale vaccine production and

10

marketing rights have been arranged, in the form of a collaboration with a vaccine manufacturer; characterization and mapping of a plasmid involved with entero-adhesiveness of enteropathogenic Escherichia coli; development of techniques to measure the T lymphocyte-mediated immune response to antigens of Salmonella typhi. Animal studies to elucidate the pathophysiological mechanisms of various enteropathogens either have been done or are in progress.

Among clinical studies underway are evaluation of the immunogenicity and protective efficacy of new cholera vaccine candidates, including several killed antigen combinations (toxoid plus killed whole vibrios), as well as attenuated V. cholerae strains used as oral vaccine; studies of immunity to cholera across biotype; evaluation of attenuated influenza virus vaccines; assessment of the immunogenicity and efficacy of an attenuated Shigella sonnei vaccine; evaluation of the immunogenicity and protective efficacy of purified CFA/II pili oral vaccine; studies of homologous and heterologous immunity in Campylobacter jejuni infection; and studies to develop strategies toward oral rehydra- tion during diarrheal episodes.

Some areas where biotechnology will be applied with good prospects for success are the following.

Influenza, a viral respiratory infection, is a major source of morbidity and mortality in Marylanders during influenza A epidemic years and, as a conse- quence, directly accounts for diminished productivity throughout the state. Influenza B infections in school children can be followed by Reye's syn- drome (hepatic dysfunction and encephalopathy) which has a high fatality. Collaborative research efforts have been underway for several years between the Center for Vaccine Development, Uni- versity of Maryland, and the Laboratory of Infectious Diseases of the National Institute of Al-

...lergy and Infectious Diseases resulting in the prep- aration and evaluation of several new live attenuated intranasal influenza virus vaccine candidates.

Rocky Mountain Spotted Fever in Maryland represents one of the highest incidences of this infection in the United States. Unless diagnosed rapidly and treated promptly, it can result in severe long- term sequelae or death. A satisfactory vaccine does not yet exist. Research on development of new

vaccines against this disease is underway at the University of Maryland. With enhanced support, investigators will apply the powerful and innovative tools of advanced biotechnology to this problem.

Enterotoxigenic Escherichia coli is a class of bacteria which represents the major etiologic agent of travelers' diarrhea affecting travelers to less-developed areas of South America, Asia and Africa. On- going research has been utilizing recombinant DNA technology, sophisticated immunologic techniques and electron microscopy to develop vaccines against this infection. Two vaccines have reached the stage of clinical testing.

Enteropathogenic E. coli is a class of bacteria which causes diarrheal disease in infants. Molecu- lar genetic studies have resulted in major revela- tions regarding the pathqgenesis of this infection, opening the way for the development of a vaccine.

Campylobacter jejuni is a bacterium associated with diarrheal disease in man and animals. The natural reservoir includes poultry and farm animals; approximately 90 percent of Maryland chick- ens are infected. Illness can be transmitted to man by handling live or uncooked chicken or by eating undercooked chicken. Studies underway involve investigation of the pathogenesis of and immunity in C. jejuni infection. The ultimate goal is a vaccine to protect against C. jejuni.

In addition, a variety of bacteria normally resi- dent in the waters of the Chesapeake Bay have recently been shown to cause infection in man. These species include V. cholerae 01 and non-01 sero- groups, V. vulnificus, V. hollisae, V. damsela, V. mimi- cus, V. fluvialis, and V. parahaemolyticus; Aeromonas hydrophila, another pathogenic member of the family Vibrionaceae, is also present in the Bay. These pathogens are acquired either by contact with waters of the Bay or by ingestion of seafood, particularly crabs. Development of vaccines for these infections has not begun because so little is known about the pathogenesis of disease due to these species. Basic research aimed at elucidating their pathogenesis is underway, a necessary first step before prophylactic measures can be developed.

The most current techniques in biotechnology are also being applied to develop rapid diagnostic techniques for certain diseases. For example, these include the use of cloned DNA gene segments as

11

hybridization probes to detect the presence of homologous genes in test specimens as well as the use of monoclonal antibodies in various immuno- logical assays to detect the presence of cell wall antigens and/or toxins. The specificity and rapidity of these methods offer great promise for speedy diagnosis of pathogenic agents.

As detailed above, multiple species of Vibrio exist in the Chesapeake Bay and are the cause of infection in man. While these infections are not believed to be common, their precise incidence is not known. Moreover, there may be considerable under-reporting of infection, mainly because of the difficulties in diagnosis. Rapid diagnostic techniques are required to identify both infections in man and contaminated seafood. The implications for industry of seafood-related outbreaks of illness are obvious.

Modern tools of biotechnology for application to diagnostic techniques include using cloned DNA segments as specific probes and monoclonal antibody techniques for the detection of pathogenic Vibrio species.

Cloned DNA sequences that encode genes for the production of E. coli heat-labile (LT) and heat- stable (ST) enterotoxins are currently in use for the rapid diagnosis of enterotoxigenic E. coli infections.

Work is underway to develop a gene probe to identify enteropathogenic £. coli from other £. coli. The diarrheagenic £. coli possess a plasmid (circa 60 Mdal in size) that contains genes involved in the ability to adhere to enterocytes. The critical genes are being cloned and characterized, thereby leading to a probe for rapid diagnosis.

Biotechnology Applications in

Agriculture and Fisheries

Since agriculture is Maryland's largest industry, biotechnology capabilities are being applied to combat animal and avian diseases whose effect could have serious economic significance. New techniques are required to prevent the economic hardships associated with these diseases which are not arrested by present immunization programs. One such disease is infectious bronchitis virus (IBV) in poultry. IBV represents a serious threat to

an industry which is a vital segment of Maryland's economy. The value of the broiler industry in 1981 exceeded $325 million and created employment opportunities on Delmarva for about 15% of the population. A 1979 estimate of broiler losses due to IBV on the Delmarva Peninsula was $6 million. Present efforts to control IBV have centered on flock vaccination. IBV outbreaks have been found to occur in both vaccinated and unvaccinated flocks. University researchers have concluded that IBV, like other major diseases, contains numerous variants and, therefore, no single vaccine can treat all forms of the disease.

To address this problem, the University's Agri- cultural Experiment Station is developing monoclonal antibodies to diagnose a host of diseases and their variants. These antibodies are produced by combining tumor cells and IBV antibody producing cells. The product of this combination is a specific antibody capable of identifying a specific form of IBV. If scientists can manufacture enough specific monoclonal antibodies to detect large numbers of the variations of animal and avian diseases, more effective diagnosis and treatment of these diseases will be forthcoming.

University scientists are

developing a diagnostic method

that will enable poultry

producers to keep a daily

account of the flock's health.

The University will enhance this pioneering effort by developing a complementary diagnostic process for poultry producers. A diagnostic method known as "flock profiling" will be developed to enable producers to keep a daily account of the flock's health. Once perfected, this technique will be described and demonstrated to the farming community through the Cooperative Extension Service, only one example of the many potential efforts envisioned under the aegis of the Maryland Biotechnology Institute.

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This program of research and extension has economic implications beyond the broiler house: other major animal industries in Maryland are hampered by diseases of economic significance. Bovine mastitis, an inflamation of the mammary gland in cows, costs Maryland dairy farmers approximately $24 million yearly through production loss, veterinary fees and drugs. The dairy industry in Mary- land accounts for over $200 million annually and is a vital part of the economic sector in western Mary- land. University researchers believe that monoclonal antibodies could lead to the development of a mastitis vaccine.

Application of this research to the state's billion dollar equine industry will yield important results. For example, the mysterious Potomac Fever, which killed 37 of the 95 stricken horses in Freder- ick, Howard and Montgomery counties, may become less mysterious if the diagnostic abilities of monoclonal antibodies are applied.

Researchers believe that monoclonal antibodies may solve major health problems hampering the growth of Maryland's existing agricultural industries. Yet rapid advances in this technology will benefit from a larger effort by the University of Maryland Biotechnology Institute. The manufacture of monoclonal antibodies, their screening for effectiveness and their accumulation as a "seed stock" against a specific disease and its variants, requires both substantial research efforts and additional support of the efforts.

Maryland's fisheries also face serious disease problems. Dealing with disease in a marine or estuarine environment will be far more difficult than dealing with disease in animal agriculture on land. Our knowledge of diseases in fisheries is limited, particularly when diseases occur in the juvenile stage. At present, several economically-significant diseases affecting our fisheries resources have been identified.

The oyster beds in Maryland have been plagued by MSX (Haplosporidium nelsoni), a tiny, spherical parasite which starves and weakens oysters by dis- rupting their metabolism and nutrition. DERMO, another parasite, can also cause significant dam- age; 1983 gives evidence of this; the oyster harvest, with reportedly high occurrences of MSX, was down 30-40 percent from 1982, when there were virtually no reports of MSX or DERMO.

The potential economic effects of disruption to the fishing industry in Chesapeake Bay, as a con- sequence of disease, can be significant. Maryland has 25,700 people employed in the industry, and the dockside value of the catch is estimated at $45 million, 50 percent of which is from oysters.

Thus, knowledge developed in efforts to contain human and animal diseases will be applied to diseases of fish. Work is currently under way on the study and analysis of our estuaries and the fauna of those estuaries. For the commercially important fisheries, a knowledge base has already been developed by scientists of the University of Maryland Sea Grant College, Agricultural Experiment Sta- tion and Medical School (UMAB) that will apply to those agents afflicting Maryland's fisheries. The biotechnology approach, then, offers an extraordinary opportunity to understand, control and even- tually attenuate or eliminate fish diseases plaguing Chesapeake Bay stocks.

Bioelectronics, Biosensors, and

Artificial Intelligence

The objectives of research in this area will include use of artificial intelligence systems in medicine, data management and studies leading to the development and application of organic molecules—biochips—in electronic circuitry.

Artificial Intelligence in Medicine

Artificial intelligence (AI) is a branch of computer science concerned with the automated manipulation of non-numeric symbols to produce computer programs exhibiting human-like intelligence. Biomedical applications include "expert systems" that simulate human medical experts (and thus serve as decision aids for patient diagnosis and treatment), models of psychiatric illnesses such as paranoia, analysis of language errors made by aphasic (brain damaged) patients, and natural language interfaces to clinical data bases.

The University of Maryland currently has a strong research program in medical AI, with one project centered on the development of mathematical and computer models of the diagnostic reason- ing process of physicians. Another project has involved the development of experimental, Eng-

13

lish-like programming languages that are suitable for direct use by physicians in building computer- based decision aids. The use of AI methods to screen prescriptions in the context of other patient information to minimize side effects and drug reactions is also in progress.

A computer-supported decision aid for evaluation of patients with transient ischemic attacks (TIAs) is under development at the Department of Neurology, University of Maryland at Baltimore and the Department of Computer Science, Univer- sity of Maryland at College Park. This system uses advanced artificial intelligence technology to simulate the expertise of stroke specialists in evaluation of TIA patients. The program uses strict criteria to classify TIA patients, localize their neurological deficit, screen for causative disorders, recommend additional tests and suggest patient management steps. The first version of this decision aid was recently tested using information on one hundred patients seen by the University of Maryland Hos- pital Stroke Service. The decision aid exhibited near-expert level performance during this preliminary trial. Based on this initial work, a second gen- eration TIA decision aid is being planned in conjunction with a more general, on-line intelli- gent textbook of neurology.

Artificial intelligence is

anticipated to be the major

growth area in computer

software during the next two

decades.

AI is currently undergoing a period of rapid advance and application, and is anticipated to be the major growth area in computer software during the next two decades. Research needs during this period include the development of new methods for computer processing of medical knowledge, parallel computer architectures that support so-

phisticated AI software, and improved interfaces for computer-inexperienced users, including many physicians.

Decision Making. Most medical decisions, including diagnoses, are based upon the frequencies with which signs and symptoms are associated with various disease states. Artificial intelligence is merely the explicit performance by machine of what is usually a less-than-explicit human process. Automated decision-making, as a component of biomathematics, will be a focus of the over-all biotechnology thrust. Areas other than medicine will be significantly advanced by application of these methods. With the well recognized strength of the University in computer science and electrical engineering, this is an area where Maryland can become an unquestioned leader.

Genetic Modeling

The interaction between genes and environment is known to be important in the determination of many traits, including several birth defects. It is often difficult to establish the relative importance of the genetic and environmental contributions, and often impossible to detect specific environmental agents associated with increased risk, or even to define the genetic determinants of disease. Genet- ic-epidemiological studies involving large samples of patients and appropriately matched controls provide some promise. Another approach with broad application is the development of computer simulation studies to investigate the outcomes of complex models of genetic-environmental interactions. Examination of the incidence, prevalence and recurrence risks for a defect under models controlled for gene frequencies, transmission probability, exposure frequency, mating patterns and family size will provide the opportunity to explore the potential of differentiating among etiol- ogies under non-controlled circumstances.

Mathematical Modelling of Physiologic Systems

Converting descriptions of biologic systems into mathematical statements permits comparisons of systems and prediction of future systems performance. A goal here will be to expand both the biologic systems described mathematically and the mathematical techniques used in those descrip-

14

tions. It is expected that physiologic system testing and understanding will be improved, ultimately leading to a better understanding of disease processes.

Adaptive Control of Biologic Systems. A natural outgrowth of modelling is adaptive control, which permits optimal system control based upon knowledge of the system (the model) and upon current system performance, that is, adaptive control permits adaptation of current controlling forces based upon system response to prior controlling forces. Biomathematics will be used to explore ways in which adaptive control techniques can be applied to management of disease states.

Bioelectronics and Biosensors

When vacuum tube electronics were replaced by transistors and then microchips, technological capability advanced with astonishing rapidity. The prospects for bioelectronics—the use of organic molecules, or biochips produced by genetically engineered bacteria—hold the promise of even greater technological possibilities. Wide-spread application of biochips is felt to be a certainty within the next three decades. Progress in this field will rely upon the creative combination of expertise in the areas of hybridoma research, artificial intelligence, surface physics, immunology and software engineering. The University of Maryland offers the unique resources of expertise in these areas as well as geographic proximity to industrial leaders in biochip production research.

Development of a functioning biochip requires several steps. A collection of antigens possessing special steric properties and monoclonal antibodies to form the backbone of the chip must first be established. The means for interconnecting these building blocks (so called moletons) in a variety of ways (stimulatory, inhibitory, permissive) must then be established, along with methods for interfacing the biochip with conventional electronic devices. While research at the University of Maryland will contribute to various phases in biochip development, special promise is seen for work in artificial intelligence and the software engineering of this new "hardware," as well as in the clinical testing of implantable biosensors and other bioelectronic prosthetic devices. The market po-

tential for this technology significantly increases the probability of the Institute's self-sufficiency over the long term.

Marine Biotechnology

Genetic engineering applied to the production of fish, molluscs and crustaceans in natural environ- ments and hatchery systems, although at the rudi- mentary stage, offers unique promise. Applied to these animals, in vitro manipulations such as cloning, cell fusion, production of chimeras and other recombinant DNA techniques will provide an im- petus for major advances in applied genetics. Suc- cessful aquaculture of many species of invertebrate animals has been achieved, since large populations of shellfish at the larval and intermediate stages can be manipulated and their genes cloned. Thus, the stage is set for the realization of genetic engineering's great potential for the marine sciences and its particular application to species in Chesa- peake Bay.

The most dramatic examples of biotechnological application are those of marine pharmaceuticals. Cardiotonic polypeptides from sea anemones, an adrenergic compound from the sponge, Verongia fistularis, and potential anticancer agents have been discovered in Caribbean gorgonians and soft corals. The tunicate of the Trididemnum genus, when extracted with methanol-toluene (3:1), showed activity against herpes simplex virus, type 1, grown in CV-1 cells (monkey kidney tissue), in- dicating that the extract inhibited the growth of the virus. This antiviral activity may also involve anti- tumor activity.

The literature describes a variety of compounds from the sea which act on the cardiovascular and central nervous systems. Drugs of high pharmaco- logic activity from nature have, in fact, been unsur- passed by synthetic compounds. Drugs from nature, predominantly from plants, include mor- phine, atropine and digitalis glycosides, to name but a few. Marine animals and plants have also yielded cardiovascular-active substances, and these include histamine and N-methylated hista- mines of sponges, asystolic nucleosides from the sponge, and the nucleoside, spongosine, isolated from Cryptotethya cn/pta.

15

Thus, only a few marine organisms are currently sources of useful drugs. Genetic engineering can change this situation dramatically, by opening up a vast and diverse range of marine life to probing for valuable pharmacological compounds. In the long run, these opportunities will develop as the

Genetic engineering can open

up a vast and diverse range of

marine life for use as valuable

pharmacological compounds.

tools for gene cloning are sharpened and the applications broadened.

Marine Toxins

Of particular interest are toxins produced by marine organisms such as that of the sea nettle in Chesapeake Bay. A toxin has the potential to be applied as a drug or pharmacological reagent. Fur- thermore, even if direct use as a drug is not feasi- ble because of potent or harmful side effects, the toxin can serve as a model for synthesis or im- provement of other drugs. Many attempts have been made to develop useful drugs from the sea by screening for anticarcinogenic, antibiotic, growth- promoting (or inhibiting), hemolytic, analgetic, antispasmodic, hypotensive, and hypertensive agents.

Marine toxins show great promise not only as pharmacological reagents, but also as models for the development of new synthetic chemicals. The need, at the moment, is for strategies for collect- ing, culturing and screening marine organisms from which bioactive agents can be isolated and characterized. Most likely, the immediate suc- cesses will ocur in discoveries of novel antibacter- ials or antibiotics produced by marine bacteria. However, the potential for engineering the production of the more complex pharmaceuticals exists. The Chesapeake Bay offers a rich source of biological material.

Industrial Chemicals

Directed search for non-antibiotically active natural products in the marine environment, especially those described earlier as being unique to marine organisms, can open an entirely new source of industrial chemicals. What is needed are new and novel screening strategies for such products. Then, the techniques of genetic engineering will remove the limitations of remote geography and the need for large harvests. Cloning genes effective for producing desired compounds in a non- marine, industrially adapted vector offers rich sources of new products.

Biodegradation in the Marine Environment

In contrast to natural products, man-made compounds are relatively refractory to biodegradation, often because naturally present organisms cannot produce enzymes necessary for transformation of the original compounds, such that the resulting in- termediates can enter into common metabolic pathways to be metabolized completely; thus, special problems are created for waste treatment and environmental protection.

Required steps to initiate biodegradation are rea- sonably well understood. Halogenated compounds, we know, are particularly persistent because of the location of the halogen atom, the halide involved and the extent of halogenation. Controlled mixed cultures are already in use in Ja- pan for treating selected industrial wastes in reac- tors. These cultures are composed of heterotrophic bacteria, photosynthetic bacteria and algae. Var- ious methods of genetic engineering will certainly become widely used to develop optimized prolif- eration and maintenance of selected populations.

What has not yet been considered, however, is the engineering of microorganisms to be added to wastes that are to be discharged into the marine environment. With increased use of the world oceans for human waste, attention must be paid to the problems of marine pollution. Pollutants enter- ing the ocean that can interfere with the integrity of ecosystems include synthetic organics, chlorina- tion products, dredged spoils, litter, artificial ra- dionuclides, trace metals and fossil fuel compounds. And the University of Maryland Bio-

16

technology Institute will be in a particularly good position to take the lead in applied research here.

Molecular biology offers great possibilities to develop biological agents that manifest enhanced resistance to industrial pollutants and that can even directly combat pollution (such as oil spills). How- ever, in order for such research to be maximized and facilitated, a host-vector system will have to be devised that allows the cloning of genes in marine organisms. Heretofore, such a system has not ex- isted. However, research is currently underway at the University of Maryland to develop such a system. A success in overcoming this hurdle will open the way to genetically engineer marine bacteria that can prevent biofouling of ships, dissolve oil slicks, combat fecal pollution, and prevent diseases in fish and man.

From another perspective, the need for algicides and antifouling agents is so great that breakthroughs in obtaining compounds with these activities will guarantee market success.

In addition, specific problems of seafood industry wastes, such as shellfish waste, have not been

Biotechnology offers

extraordinary opportunities for

aquaculture.

considered from the viewpoint of engineering microorganisms to biodegrade the wastes rapidly, even though conversion of the shellfish waste, chitin, to single-cell protein has been considered. Ap- plication of genetic engineering to improved food yield is clearly a very promising area upon which to focus.

The industry developed to exploit chitin or its derivatives remains small. Two companies in the United States produce chitosan, and there is some production and marketing of chitosan in Japan. This area of research, i.e., genetic engineering applied to pollution technology, should follow quick- ly on the heels of any breakthroughs occurring in waste treatment processing.

Bioengineering can bring big payoffs for aquaculture. Marine microorganisms offer new sources of biomass and represent a major opportunity for the future. In the United States, most of the tradi- tional fisheries are being harvested at or near maximum sustainable yields. Approximately sixty percent of the fisheries products consumed in the United States are imported, representing a trade deficit in excess of $2.5 billion. Mariculture offers the potential for reducing this deficit. Exploiting microbial sources of protein at the larval stages and during larval metamorphosis and growth can reap enormous benefits and profit.

That aquaculture itself can pay off is already established. China, for example, produces two million metric tons of finfish every year, mostly in the form of carp grown in ponds, lakes, reservoirs and ditches. In the United States, interest in aquaculture is on the rise, and that interest has meant increases in our knowledge of marine biology and the technical expertise to apply discoveries in marine biology to aquaculture. The University of Maryland has the opportunity to take the lead in applying these sophisticated, more efficient methods for aquaculture.

A major problem of aquaculture is disease, predominantly microbially mediated infections and epidemics. Viral and bacterial agents that are common hatchery complaints include IPN, as noted above, egtved and other viruses, and Vibrio and Aeromonas among the bacteria. Many causes of disease and loss of hatchery stocks are still not yet known, nor are controls of epizootics yet available. An extract from Ecleinascidia turbinada (Ete) has been shown to enhance the hemocyte function of invertebrates, including the blue crab (Callinectes sapidus), possibly rendering the animals more resistant to infection.

Thus, biotechnology offers extraordinary opportunities for aquaculture. Many species of shellfish and finfish are available in culture, providing excellent opportunities for selection and gene manipulation. Production, stabilization and delivery of vaccines, employing both hybridoma technology and genetic engineering, will enhance productivity from the egg through the larval stages, presently a high-risk portion of the life cycle.

Stock assessments of migrating fish and species

17

identification remain unresolved issues in fishery management. A method for comparison of mitochondrial DNA (mt DNA) from different individ- uals offers an opportunity for mapping the mt DNA genome and is being explored by several investigators. The possibility of using genetic mark- ing, by introducing selected genetic traits into fish, as opposed to mechanical tags, is yet another ave- nue for genetic engineering in aquaculture. Fish wander over long distances without barriers to their movements, but there may now be a way to detect subpopulations or specific independent stocks by very precise methods yielding unequivo- cal results.

An important research program at the Universi- ty of Maryland is focusing on the genetic engineer-

Maryland researchers have

identified a new bacterium

associated with the settlement

of oyster larvae, a finding that

will have significant

implications for shellfish

production.

ing of the process of molluscan larvae attachment to surfaces. Efforts are concentrated on the process affecting the life stages of molluscs in the transition from free swimming larvae to sessile "spat." A bacterium called LST has been isolated from the American oyster, Crassostrea virginica. Since the isolation of LST four years ago, the bacterium has been thoroughly characterized and found to represent a new bacterial genus and species. LST synthesizes two important exopolymers, the first of which is a melanin. Maryland researchers have shown that under various conditions one or more products of the chemical pathway that produces melanin attracts oyster larvae and promotes their settlement. The researchers are now in the process of "shotgun" cloning the LST gene pool, using a technique that will permit transfer of the genes to a

number of procaryotic systems. In essence, biotechnology methods are providing us the means to identify and focus on the compound(s) that affect oyster settlement which will have significant implications for shellfish production.

The second exopolymer synthesized by LST is a highly viscous slime layer that facilitates strong adhesion by the oyster larvae. The working hypothe- sis is that LST is chemotactically attracted to a surface, reversibly adheres by means of a micro- capsule, synthesizes invertebrate attractants and protective chemicals, and encases an entire macro and micro community in a viscous permeable layer of slime. The control mechanisms and biosynthetic pathway of this second exopolymer synthesized by LST are being elucidated using recombinant DNA technology.

University scientists have observed that the bacterial system found to operate in Chesapeake Bay and enhancing the attraction, settlement and morphogenesis of oyster larvae may also be active in a group of marine procaryotes that have been discovered in the hydrothermal vents, located near the Galapagos Islands, at a depth of 2500 meters. From a single mussel-like animal found at the hydrothermal vents, several bacterial species of the genus Hyphomoms have been isolated. These bacteria also synthesize adhesion polymers and melanin, an intriguing aspect of these bacteria. The tolerance of Hyphomoms to a wide range of envi- ronments is now under examination. Because these bacteria thrive at one atmosphere, as well as at the high hydrostatic pressures at great depths in the ocean, they also have envelopes that are more resistant to changes in pressure than those of terrestrial procaryotes. These deep-sea bacteria are being examined as potential recombinant DNA hosts for expression of terrestrial genes in the marine environment. The possibility exists that Hy- phomoms may be useful in synthesizing isoenzymes that are functional under a specific set of environmental conditions.

Thus, Maryland researchers now have exciting results stemming from basic research that have practical applications. Foremost is the discovery that neurotransmitter substances produced by estuarine and marine bacteria serve to attract molluscan larvae to surfaces and enhance larval development and morphogenesis. The bacterium

18

associated with settlement of Crassostrea virginica is now being patented. Clearly, this is but one of many new, exciting technologies to be developed in marine biology. The process that has been elucidated can result in a marketable larval attractant and morphogenesis inducer. More importantly, the discovery of a neurotransmitter that is produced by an estuarine bacterium and affects larval settlement and development opens an exciting area for further research on basic and fundamental mechanisms in nature.

Another active marine biotechnology research

project is represented by the work at The Johns Hopkins University, which is being supported by the University of Maryland Sea Grant College. In certain finfish, genes are expressed in response to environmental parameters. These gene families include the "antifreeze gene" complex, and the me- tallothionein gene complex, and they are expressed in response to environmental temperature and metals, respectively.

Fifteen years ago it was discovered that Antarc- tic fishes were able to keep from freezing in salt water at temperatures of -20C by synthesizing a polypeptide that contained periodic saccharides. These proteins depressed the freezing point of the fish fluids, but not the melting point. The structure of the first antifreeze protein was found to contain a sequence of alanine, threonine, proline and poly- saccharides. Since Arctic and Antarctic fish contin-

ually experience low temperatures, the antifreeze genes are always "turned on." However, fish living in temperate climates, such as the winter flounder Pseudopleuronectes americanus, experience cold water only on a seasonal basis. It was found that the antifreeze proteins discovered in Arctic and Antarctic fishes were present in winter flounder in the cold months, but absent in the summer. The synthesis of these proteins, in vivo, was found to be correlated with temperature and photoper- iod. More specifically, few proteins, other than antifreeze proteins, are synthesized in the winter flounder during the winter and spring (November to April). During the summer no significant levels of antifreeze mRNA can be detected. The appear- ance and disappearance of the mRNA is also correlated with seasonal changes in antifreeze protein in the serum.

The antifreeze system is an excellent model for studying how environmental parameters effect gene expression through whole organism and cellular events. By using exogenous gene transfer methods, we may be able to provide unrelated organisms with the antifreeze capability. The practical application of this research is that it may also be used for low temperature cellular and/or organ storage or for providing antifrost protection for plants.

Another area in which basic research in marine biotechnology could provide tremendous benefit concerns the development of strains of marine bacteria capable of biodegrading toxic compounds, such as 2,4,5-T, hydrocarbons and other, even more complex (and recalcitrant), compounds en- tering the marine environment. At present, disposal at sea is done without inoculation of wastes to be dumped in the ocean with marine bacteria which can degrade those materials under ocean environmental conditions. Clearly, marine bacteria need to be studied as the source and host for genes that can enhance degradation of material discharged into the ocean. Bacteria biodegrading wastes in sewage plants can be applied to terrestrial waste treatment but not for disposal of waste at sea, because terrestrial bacteria do not function well (if at all) in seawater. This is a research area which the expertise of the Maryland Biotechnology Institute would be well equipped to direct.

One more application of biotechnology relates to

19

fisheries management. Mitochondrial mt DNA se- quencing provides a detection device for determining the origins of fish. That is, by mt DNA sequence pattern analysis, it should be possible, in the next few years, to determine from what estuary fish such as striped bass derive, and even the sub-estuary or tributaries of the estuary that they come from. The application of mt DNA analysis to salmonid species, and other fishes of the high seas, could provide an extraordinary method for determining, without ambiguity, the origins of the fish in a catch, hence the "national source" of the fish. Preliminary research in this area is underway through the Maryland Sea Grant College.

Scientists at the University have

shown that monoclonal

antibodies can be developed

successfully to protect fish

against viral and bacterial

infections.

the purely descriptive stage to the highly quantitative, analytical stage of science.

Biophysical Technology—A Cooperative Program

The alliance between the University of Maryland and the National Bureau of Standards goes back decades. It has been especially strengthened in recent years because of the enthusiasm for interaction by leaders of these institutions and because steady-state budgets have encouraged collaboration to make funding go further.

The campuses of the University of Maryland at College Park, Catonsville and Baltimore all house many research laboratories in which biotechnology can be found. The nature of this research ranges from agriculture to pediatric medicine. Coinciden- tally, the National Bureau of Standards has initiat- ed in its National Measurement Laboratory a life sciences group. This group is primarily concerned with the problems stated above, namely that analytical techniques must be brought to bear on the largely qualitative methods which are currently important in biotechnology.

Hybridoma technology has also been applied in fisheries science to develop fish vaccines. At the University of Maryland, investigators have shown that monoclonal antibodies can be developed successfully to protect fish against viral and bacterial infections. These developments will enhance the capability of rearing fish in closed systems, as well as improve the economics of fish and shellfish culture. More importantly, some of the studies of these highly pure antibodies may elucidate the viral pathology of related infections in humans. There appears to be an intriguing relationship between the infectious pancreatic necrosis virus activity in fish and that of the onset of juvenile diabetes in humans. Clearly, the application of hybridoma and monoclonal antibody technology to the marine sciences will provide some fascinating developments in basic marine biology with "spill- over" for human health. Thus, by making use of biotechnology, marine biology can be taken from

As described above, the "biology" components of biophysical technology are located in many departments on the University of Maryland campuses at College Park, Catonsville and Baltimore. Most areas of study in the fields of biochemistry

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and physiology now use the techniques of biotechnology, including gene splicing and the use of hy- bridomas. The "physics" activities are located, in large part, at the National Bureau of Standards. Research in the bioanalytical and clinical chemistry is taking place at the National Bureau of Stan- dards, the Center for Chemical Physics, the Center for Analytical Chemistry, the Center for Radiation Research and in the various inter-Center Health Measurements Programs. A recent meeting of the major administrators of the University of Mary- land and the National Bureau of Standards has made it clear that the biophysical technology program, supported by the NBS/University of Mary- land collaborations, will be an important component of the Maryland Biotechnology Insti- tute. Areas of collaboration include the following:

• Biothermodynamics, measurements of ther- modynamic properties of biologically important systems such as hexokinase reactions, bacterial growth, mitosis of sea urchin eggs. Additionally, a microcalorimetry laboratory has been set up to measure enthalpy changes and equilibrium constants of enzyme cata- lyzed reactions. Also, a biochemical prepara- tion laboratory is being set up to prepare, purify and characterize enzymes.

• Quantum Chemistry and Enzymatic Mecha- nisms. The goal of this research is to determine enzymatic mechanisms by theoretical calculations of the electronic structure and properties of appropriate models of the active site of the enzyme. The biochemistry approach to discover the mechanism and func- tions of the enzyme invariably starts with a determination of the structure. The quantum chemistry approach would extend this initial step to an understanding of the electronic structure. Computational capabilities have been developed by the Quantum Chemistry Group which will allow us to understand enzymatic reactions from the point of view of the enthalpy changes in the local active site environment.

• Qualitative/Quantitative Analyses of Biomole- cules, which includes two-dimensional elec- trophoresis (2-DE) to establish optimal gel chemical composition and operative parameters to effect reproducible separations; pro-

tein marker series for "retention indices"; purity of vaccines; patterns of bacterial mem- branes and specific fish species as pollution markers.

• Mechanisms of Metal Ion-Protein Interactions, for developing specific, sensitive staining techniques for determining protein locational information of 2-DE gels; thin layer isoelectric focussing to increase separation efficiency and decrease separation time; and high performance liquid chromatography of peptides, steroids and other biomolecules.

• Use of Biomolecules as Analytical Reagents to develop chromatographic techniques (metal ion affinity chromatography for protein separations based on coordination chemistry principles), and the use of enzymes for activity measurements and for substrate analyses are among the goals of the program.

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Summary

The benefits to the State of Maryland from the proposed center of excellence in biotechnology at the University of Maryland

are both direct and indirect. They include development of vaccines that prevent diseases of importance to Marylanders both at home and when they travel abroad, development of rapid diagnostic methods for combatting diseases of importance to citizens of the state and nation, and production of diagnostic techniques that will allow seafood to be economically screened for the existence of potential pathogens. Ad- vances in plant biotechnology could rev- olutionize agriculture. These are but a few examples of the potential of this exciting, new development in science and technology.

The economic well-being of the State of Mary- land will be enhanced by providing resources for the University to interact with the already strong nucleus of biotechnology firms in the state. Tech- nical support, technical service, cooperative work and joint undertakings are but a few of the areas for University/industry interaction. Furthermore, in strengthening the University of Maryland as a center of excellence in biotechnology, educational opportunities at all levels within the state will be available. The Maryland Biotechnology Institute will address directly undergraduate and graduate education, training of technicians, and continuing education, and will advance the knowledge and

application of fermentation processes and bench and pilot skills. Exciting education and research opportunities will be made available locally, draw- ing on the resources of the University of Maryland. All campuses of the University, by encouraging collaboration among investigators, will make the

sum greater than the constituent parts, in productivity and creativity.

Biotechnology developments within the Univer- sity of Maryland, without question, will prove an asset for the State of Maryland. Past contributions of the University of Maryland to the economic well-being of the state have been well recognized, but a truly outstanding state University will attract new industry by the atmosphere created when tal- ented, creative and dynamic teachers and researchers are brought together in a center of excellence.

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university of maryland task force on high technology

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