It is delightful but humbling to find my face at the start of these Proceedings--there are innumerable other faces which could equally weIl stand there, from among the band who have fore gathered at every gerontology conference since the subject was launched in its present form; but I deeply appreciate being there. Gerontology d. id not grow by accident. Its present standing is the fruit of careful planning, undertaken by European and American scientists back in the 1950's. In those days it was still a "fringe" science, and the conspirators had much the standing of the 1920's Interplanetary Society. The United States itself is the offspring of conspiracy, for when the results of conspiracy are beneficent, the conspirators become Founding Fathers. This has been the case with gerontology. The present meeting is especially gratifying because the papers have been recitals of normal, hard-science investigation. We had to get through the rigors of a long period of semantic argument and a long period of one-shot general theories before this kind of meeting, normal in all other research fields, could take place. It was also necesssary to breed in the menagerie a generation of excellent investigators aware of the theoretical background but unintimidated by it, who share our conviction that human aging is comprehensible and probably controllable, and who go into the laboratory to attack specifics."

This symposium is the third in a series featuring the propaga tion of higher plants through tissue culture. The first of these symposia, entitled "A Bridge Between Research and Application," was held at the University in 1978 and was published by the Technical Information Center, Department of Energy. The second symposium, on "Emerging Technologies and Strategies," was held in 1980 and pub lished as a special issue of Environmental and Experimental Botany. One of the aims of these symposia was to examine the current state of-the-art in tissue culture technology and to relate this state of technology to practical, applied, and commercial interests. Thus, the third of this series on development and variation focused on embryogenesis in culture: how to recognize it, factors which affect embryogenesis, use of embryogenic systems, etc.; and variability from culture. A special session on woody species again emphasized somatic embryogenesis as a means of rapid propagation. This volume emphasizes tissue culture of forest trees. All of these areas, we feel, are breakthrough areas in which significant progress is expected in the next few years."

It is delightful but humbling to find my face at the start of these Proceedings--there are innumerable other faces which could equally weIl stand there, from among the band who have fore gathered at every gerontology conference since the subject was launched in its present form; but I deeply appreciate being there. Gerontology d. id not grow by accident. Its present standing is the fruit of careful planning, undertaken by European and American scientists back in the 1950's. In those days it was still a "fringe" science, and the conspirators had much the standing of the 1920's Interplanetary Society. The United States itself is the offspring of conspiracy, for when the results of conspiracy are beneficent, the conspirators become Founding Fathers. This has been the case with gerontology. The present meeting is especially gratifying because the papers have been recitals of normal, hard-science investigation. We had to get through the rigors of a long period of semantic argument and a long period of one-shot general theories before this kind of meeting, normal in all other research fields, could take place. It was also necesssary to breed in the menagerie a generation of excellent investigators aware of the theoretical background but unintimidated by it, who share our conviction that human aging is comprehensible and probably controllable, and who go into the laboratory to attack specifics."

Gilbert S. Omenn Dean, Public Health and Community Medicine University of Washington Seattle, Washington 98195 On behalf of the University of Washington, the City of Seattle, the sponsors and donors, and my co-organizers, I am delighted to welcome all of you to this Conference on Genetic Control of Environ mental Pollutants. My only regret is that Dr. Alexander Hollaender, who has inspired so many of us as young scientists and stimulated so many trail-blazing conferences in environmental sciences and in gen etic engineering, is ill and was unable to make the trip to Seattle. He sends his warm good wishes for an outstanding meeting and a fine volume. The purpose of this Conference is to identify and assess strat egies for more effectively and safely managing wastes and toxic sub stances in the environment, in part through use of genetically engi neered microorganisms. There is a sense of desperation in our soci ety that modern technologies have introduced a bewildering array of potential hazards to human health and to our environment. There is an accompanying sense of frustration that our prodigious basic re search capabilities and our technological ingenuity have not yielded practical ways to control many pollutants and waste streams, or- better still--to convert them to useful products.

This symposium is the third in a series featuring the propaga tion of higher plants through tissue culture. The first of these symposia, entitled "A Bridge Between Research and Application," was held at the University in 1978 and was published by the Technical Information Center, Department of Energy. The second symposium, on "Emerging Technologies and Strategies," was held in 1980 and pub lished as a special issue of Environmental and Experimental Botany. One of the aims of these symposia was to examine the current state of-the-art in tissue culture technology and to relate this state of technology to practical, applied, and commercial interests. Thus, the third of this series on development and variation focused on embryogenesis in culture: how to recognize it, factors which affect embryogenesis, use of embryogenic systems, etc.; and variability from culture. A special session on woody species again emphasized somatic embryogenesis as a means of rapid propagation. This volume emphasizes tissue culture of forest trees. All of these areas, we feel, are breakthrough areas in which significant progress is expected in the next few years."

William C. Taylor Department of Genetics University of California Berkeley, California 94720 It is evident by now that there is a great deal of interest in exploiting the new technologies to genetically engineer new forms of plants. A purpose of this meeting is to assess the possibilities. The papers that follow are concerned with the analysis of single genes or small gene families. We will read about genes found within the nucleus, plastids, and bacteria which are responsible for agri culturally important traits. Given that these genes can be isolated by recombinant DNA techniques, there are two possible strategies for plant engineering. One involves isolating a gene from a cultivated plant, changing it in a specific way and then inserting it back into the same plant where it produces an altered gene product. An example might be changing the amino acid composition of a seed pro tein so as to make the seed a more efficient food source. A second strategy is to isolate a gene from one species and transfer it to another species where it produces a desirable feature. An example might be the transfer of a gene which encodes a more efficient pho tosynthetic enzyme from a wild relative into a cultivated species. There are three technical hurdles which must be overcome for either strategy to work. The gene of interest must be physically isolated.

There is a time in scientific research when a number of developments coincide making it possible to progress with a tough and complicated problem. It is believed that such a time has come in the area of biological nitrogen fixation. A better understanding of photosynthesis, cell hybridization, plasmid, and gene transfer between cells not necessarily genetically related, have opened new avenues of research. New developments in traditional genetics, cell biology, biochemistry, including enzyme chemistry, and plant physi ology have brought about the feeling this is a most appro priate time to pull together the different approaches in a conference where the lines of research could be discussed and thus help to speed up developments in this area. What makes biological nitrogen fixation especially im portant is the promise that a good understanding of the basic problem would help us to make organisms more amenable to fix nitrogen, not only in symbiosis with legumes, but also with other plant species and develop a wider variety of organisms with the ability to fix N * It will also 2 encourage a search for naturally occurring N2 fixing organ isms other than the traditional N2 fixers. Some success has already been encountered in this area. Success in broadening the field of nitrogen fixing would help to increase food supply, especially in de veloping countries which cannot afford to purchase synthetic nitrogen sources.

In August. 1982. a conference was held at the University of Califor nia. Davis. to discuss both molecular and traditional approaches to plant genetic analysis and plant breeding. Papers presented at the meeting were published in Genetic Engineering of Plants: An Agricultural Perspective. A second conference. entitled "Tailoring Genes for Crop Improvement." spon sored by the UC-Davis College of Agricultural and Environmental Sciences and the College's Biotechnology Program. was held at Davis in August. 1986. to discuss the notable advances that had been made during the intervening years in the technology for gene modification. transfer. and expression in plants. This volume contains papers that were presented at this meeting and provides readers with examples of how the new experimental strategies are being used to gain a clearer understanding of the biology of the plants we grow for food and fiber; it also discusses how molecular biology approaches are being used to introduce new genes into plants for plant breeding programs. We are grateful to the speakers for their excellent presentations for the conference and extend our sincere thanks to those who contributed manuscripts for this volume."

Delbert M. Shankel Departments of }1icrobiology and Biochemistry The University of Kansas Lawrence, Kansas 66045 Welcome to the "International Conference on Mecha. nisms of Antimutagen- esis and Anticarcinogenesis. " We are delighted that so many of you have chosen to attend this first meeting on this important topic. The significance of genetic changes in cells has been recognized for many years. The seminal observations of Henri in 1914 (UV), Muller in 1927 (X-rays), and Auerbach in 1946 (chemical agents) established the fact that physical and chemical agents which may be present in our environment are capable of producing profound changes in heredity. It is now well-estab- lished, of course, that such changes can result in the development of can- cer, produce hereditary birth defects, alter microorganisms to cause drug resistance, or other harmful (or even beneficial) changes; it is likely that the processes of mutagenesis and the intricate balance between muta- genesis and antimutagenesis are involved in aging, evolution, and other fundamental life processe8. Consequently, we hope and believe that assem- bling thi. s group of scientists to share current fundamental and applied research in these areas will lead to a better understanding of these proc- esses and to long-term benefits for society. As stated clearly by Garfield (4), "Almost every aspect of modern liv- ing exposes us to health risks.

The normal course of most biologically catalyzed processes is tightly regulated at the genetic and physiological levels. The regulatory mechanisms are diverse, sometimes redundant, and it is becoming increasingly apparent that, at the genetic level, the range of mechanisms may be limited only by the permutations and combina tions available. For each microbial cell, evolution appears to have resulted in maximized advantage to that cell, achieving regulatory balance. Genetic engineering encompasses our attempts to perturb the genetic regulation of a cell so that we may obtain desired other than normal outcomes, such as increased product formation, or new product formation. Following the groundwork established by a preceding symposium (Trends in the Biology of Fermentations for Fuels and Chemicals, Brookhaven National Laboratory, December 1980), the initial planning for this conference envisioned the juxtaposition of molecular genetic expertise and microbial biochemical expertise. The resultant interaction should encourage new and extended ideas for the improve ment of strains and for the generation of new regulatory combina tions to enhance microbial chemical production from cheap and abundant (including waste) substrates. The interaction should also demonstrate that new discoveries at the basic level remain essential to progress in genetic engineering. New genetic regulatory combina tions require new studies of physiology and biochemistry to assure understanding and control of the system. New biochemical reactions necessitate new studies of genetic and regulatory interaction."

The best protection against environmental mutagens is to identify them before they ever come into general use. But it is always possible that some substance will escape detection and affect a large number of persons without this being realized until later generations. This article considers ways in which such a genetic emergency might be promptly detected. A mutation-detecting system should be relevant in that it tests for effects that are as closely related as possible to those that are feared. It should be sensitive enough to detect a moderate increase in mutation rate, able to discover the increase promptly before more damage is done, responsive to various kinds of mutational events, and designed in such a way as to maxi mize the probability that the Gause of an increase can be found. Methods based on germinal mutation necessarily involve enormous numbers of persons and tests. On the other hand, with somatic mutations the individual cell becomes the unit of measurement rather than the in dividual person. For this reason, I think that somatic tests are preferable to germinal tests, despite the fact that it is germinal mutations which are feared.

The growing concern about where energy rich chemicals for the future will come from has stimulated a resurgence of interest in the potentialities of microbial fermentations to assist in meeting anti cipated demands for fuels and chemicals. While much attention has been given recently to the early deployment of alcohol production plants and similar currently available technologies, the potential future developments have received much less attention. One of the intentions of the present symposium was to look ahead and try to perceive some of the prospects for future fermentation technology. In order to accomplish this, a symposium program of sizable diversity was developed with workers giving a representative cross section of their particular specialty as an indicator of the status of basic information in their area. In addition, an attempt was made to elicit from the various participants the types of fundamental infor mation which should be generated in the coming years to enable new fermentation technology to proceed expeditiously. In organizing the symposium particular effort was made to involve workers from the academic, industrial and governmental scientific communities."

Allen I. Laskin Biosciences Research Exxon Research and Engineering Company Linden, New Jersey I was contacted in the Fall of 1981 by Professors Martin Dworkin and Palmer Rogers, of the University of Minnesota and asked to participate in the orgnization of the 1983 conference in the series, "Interface Between Biology and Medicine." They and the other members of the advisory committee had the vision to realize that this was a time to depart somewhat from the traditional theme, since one of the major areas of interest in the biological and related sciences these days is that of biotechnology in a broader sense than its impact on medicine alone. In designing the format of the Conference, we considered another factor. There has been a plethora of conferences, symposia, and meetings on biotechnology over the past few years, and the faces and topics have become rather familiar. There has been a strong emphasis on the development of the technology and the "biotechnology industry"; less attention has been paid to the science behind it. One might get the impression from some of these meetings and from the popular press that biotechnology has just recently sprung up, apparently full blown; the very fundamental scientific discoveries and the great body of 1 ALLEN I. LASKIN 2 continuing research that forms that basis for the technology is often obscured.

This volume is the first of a series concerning a new tech nology which is revolutionizing the study of biology, perhaps as profoundly as the discovery of the gene. As pointed out in the introductory chapter, we look forward to the future impact of the technology, but cannot see where it might take us. The purpose of these volumes is to follow closely the explosion of new tech niques and information that is occurring as a result of the newly acquired ability to make particular kinds of precise cuts in DNA molecules. Thus we are particularly committed to rapid publication. Jane K. Setlow Alexander Hollaender v INTRODUCTION AND HISTORICAL BACKGROUND 1 Maxine F. Singer CLONING OF DOUBLE-STRANDED cDNA . . 15 Argiris Efstratiadis and Lydia Vi11a-Komaroff GENE ENRICHMENT . . . . . . . 37 M. H. Edgell, S. Weaver, Nancy Haigwood and C. A. Hutchison III 51 TRANSFORMATION OF MAMMALIAN CELLS . . . . . M. Wig1er, A. Pe11icer, R. Axel and S. Silverstein CONSTRUCTED MUTANTS OF SIMIAN VIRUS 40 73 D. Short1e, J. Pipas, Sondra Lazarowitz, D. DiMaio and D. Nathans STRUCTURE OF CLONED GENES FROM XENOPUS: A REVIEW 93 R. H. Reeder TRANSFORMATION OF YEAST 117 Christine lIgen, P. J. Farabaugh, A. Hinnen, Jean M. Walsh and G. R. Fink THE USE OF SITE-DIRECTED MUTAGENESIS IN REVERSED GENETICS 133 C. Weissmann, S. Nagata, T. Taniguchi, H. Weber and F. Meyer AGROBACTERIUM TUMOR INDUCING PLASMIDS: POTENTIAL VECTORS FOR THE GENETIC ENGINEERING OF PLANTS . 151 P. J. J. Hooykaas, R. A. Schi1peroort and A."

The best protection against environmental mutagens is to identify them before they ever come into general use. But it is always possible that some substance will escape detection and affect a large number of persons without this being realized until later generations. This article considers ways in which such a genetic emergency might be promptly detected. A mutation-detecting system should be relevant in that it tests for effects that are as closely related as possible to those that are feared. It should be sensitive enough to detect a moderate increase in mutation rate, able to discover the increase promptly before more damage is done, responsive to various kinds of mutational events, and designed in such a way as to maxi- mize the probability that the Gause of an increase can be found. Methods based on germinal mutation necessarily involve enormous numbers of persons and tests. On the other hand, with somatic mutations the individual cell becomes the unit of measurement rather than the in- dividual person. For this reason, I think that somatic tests are preferable to germinal tests, despite the fact that it is germinal mutations which are feared.

The ready acceptance and wide demand for copies of the first two volumes of Chemical Mutagens: Principles and Methods Jar Their Detection have demon strated the need for wider dissemination of information on this timely and urgent subject. Therefore, it was imperative that a third volume be prepared to include more detailed discussions on techniques of some of the methods that were presented from a theoretical point of view in the first two volumes, and to update this rapidly expanding field with current findings and the new developments that have taken place in the past three years. Also included is a special chapter by Dr. Charlotte Auerbach giving the historical background of the discovery of chemical mutagenesis. Methods for recognizing mutagenic compounds in vitro are a necessary preliminary step toward arriving at satisfactory solutions for recognizing significant mutation rates in man, which must be done before our test tube methods of detection can be considered reliable. Two chapters in this volume make important contributions to this problem. Due to the increasing activity in efforts to perfect techniques for detecting chemical mutagens and their effects on man, it is planned to continue this series of volumes as necessary to keep abreast of current findings.

As editor I want especially to thank Dr. Ernst Freese for helpful co operation in preparing these volumes, and to express my appreclatlOn to Drs. Kurt Hirschhorn and Marvin Legator, the other members of the editorial board. Alexander Hollaender January 1971 Preface The purpose of these volumes is to encourage the development and ap plication of testing and monitoring procedures to avert significant human exposure to mutagenic agents. The need for protection against exposure to possibly mutagenic chemicals is only now coming to be generally realized. The recently issued Report of the Secretary's Commission on Pesticides and Their Possible Effects on Health (the Mrak Report-U.S. Department of Health, Education and Welfare, December 1969) has made an important start. Its Panel on Mutagenicity recommends that all currently used pesticides be tested for mutagenicity in several recently developed and relatively simple systems. Whether recommendations such as these are actually put into effect will depend on convincing government, industry, and the public that the problem is important, that the proposed tests would be effective, and that they can be conducted at a cost that is not prohibitive. Why is it important to screen environmental agents for mutagenic activity? To those who will read this book, the answer is self-evident. The sine qua non of all that we value and all that we are is our genetic heritage.

There are many areas on this world which might lend themselves to agricultural development and which are, at the present, not used for this purpose. Two of the most obvious are desert areas where the salt concentration is very high, both land and water areas. With the development of new approaches and careful research, considerably more productive capability could be developed in these. This volume points out some of the possible approaches as well as results ob tained by a combination of creative research, practical understanding of the problems involved and inventive ways to overcome some of the handicaps of utilizing biosaline areas. This volume grew out of the "International Workshop on Biosaline Research" organized by Mr. Gilbert Devey of the Division of Interna tional programs of the National Science Foundation and directed by Dr. Anthony San Pietro of the Department of Biology of Indiana Uni versity. Since the proceedings of the workshop appeared somewhat limited, it was thought to broaden the spectra of chapters and in clude several topics briefly discussed at the Kiawah workshop."

Volume 9 of Chemical Mutagens consists mainly of chapters discussing the development and validation of short-term assays to detect the mutagenic effects of environmental chemicals. These chapters include an assay with the grasshopper neuroblast, a comparison of mutagenic responses of human lung-derived and skin-derived diploid fibroblasts, a forward-mutation assay in Salmonella, a multigene sporulation test in Bacillus subtilis, a specific locus assay in mouse lymphoma cells, a study of the induction of bacteriophage lambda, and the granuloma pouch assay. In addition, there are two chapters on the identification of mutagens in cooked food and in human feces. Frederick 1. de Serres Research Triangle Park, North Carolina vii Contents Chapter 1 The Grasshopper Neuroblast Short-Term Assay for Evaluating the Effects of Environmental Chemicals on Chromosomes and Cell Kinetics 1 Mary Esther Gaulden, Jan C. Liang, and Martha J. Ferguson 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2. Embryo Supply. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. 1. Species. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. 2. Origin of Colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2. 3. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2. 4. Colony Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . 6 2. 5. Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2. 6. Allergy to Grasshoppers . . . . . . . . . . . . . . . . . . . . . . 14 3. Grasshopper Egg, Embryo, and Cells . . . . . . . . . . . . . . . . . 14 3. 1. The Egg Shell and Membranes . . . . . . . . . . . . . . . . . 14 3. 2. Embryonic Development . . . . . . . . . . . . . . . . . . . . . . 17 3. 3. Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 4. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4. 1. Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4. 2. Preparation of Embryos for Cell Analysis . . . . . . . . . 34 4. 3. Analysis of Mutagen Effects. . . . . . . . . . . . . . . . . 40 . . . 5. Response of the Grasshopper Neuroblast to Mutagens . . . . 50 5. 1. Reproducibility of Data . . . . . . . . . . . . . . . . . . . . . . . 50 5. 2. Radiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5. 3. Chemical Mutagens . . . . . . . . . . . . . . . . . . . . . . . . . .

Additional Editors Are Arthur W. Pollister And Lewis J. Stadler. Contributing Authors Include Robert Livingston, L. J. Buttolph, J. A. Sanderson And Others.

Additional Editors Are Hermann J. Muller And Lauriston S. Taylor. Contributing Authors Include Ugo Fano, L. D. Marinelli, L. S. Taylor And Others.

Contributing Authors Include Rufus Lumry, Henry Eyring, John R. Platt And Others.

Additional Editors Are Hermann J. Muller And Lauriston S. Taylor. Contributing Authors Include Ugo Fano, L. D. Marinelli, L. S. Taylor And Others.