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New Technologies and Instrumentation Many of the recent advances in biology have been driven by the development of new technologies and instrumentation, such as recombinant DNA techniques, monoclonal antibody techniques, and microchemical instrumentation. Each of these technologies has opened new opportunities to explore both fundamental and applied biological problems. Moreover, these technologies have proven to be synergistic-each operating in conjunction with the others to amplify their poten- tials. RECOMBINANT DNA TECHNIQUES Recombinant DNA Techniques Permit Us to Isolate a Single Gene from the Tens of Thousands Encoded in a Complex Genome After a gene has been isolated by recombinant DNA techniques, studies of its structure, regulation, and function can begin. These techniques depend on the molecular complementarily of DNA molecules, which are the backbones of chromosomes and the dictionary of the genetic code, and on the two categories of enzymes that can manipulate DNA molecules. The DNA molecule is composed of two strands, each made up of a linear chain of four different building blocks: guanine (G), cytosine (C), adenosine (A), and thymine (T) (Figure 2-1~. The DNA chains exhibit molecular complementar- ity in that the Gs on one strand always pair with the Cs on the other; likewise, the As always pair with the Ts. This molecular complementarily means that in a mixture of unpaired DNA fragments, one fragment will always be able to find its complement by virtue of the precise pairing of their nucleotide bases. For this 19

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20 OPPORTUNITIES IN BIOLOGY - __ ~_ DNr~A is ~ double A_ strc~n~d helix. The ~_ r~ro strar~C. c~re con~ nected by the chem~cal _~ b~ucres A7 C, G and T: _1 A pa~rs with T; G pairs _ dSlir~ ~ A ~,~,, ;c ~ c~~ _~- w~ ~_, =~ <~ ~ cc ~ OCR for page 19
NEW TECHNOLOGIES AND INSTRUMENTATION 21 reason, a small fragment of a gene can be used to find the complete gene in a complex mixture of DNA fragments. A unique set of three contiguous DNA bases specifies one particular amino acid subunit. Given the DNA sequence of a gene, we can predict the order of amino acids in the protein it encodes. Conversely, given the amino acid sequence of a protein, one can, with certain ambiguities, predict the DNA sequence of the gene encoding it. The ability to use the genetic code dictionary to go from genes to proteins or proteins to genes is of fundamental importance to certain recombi- nant DNA strategies. Two categories of enzymes have played a critical role in the development of recombinant DNA techniques DNA-cutting enzymes, or restriction endonu- cleases, and DNAjoining enzymes, or ligases. The DNA restriction enzymes cut double-stranded DNA at precise short DNA sequences (Figure 2-2~. Thus, they provide a means of taking a large DNA molecule and cutting it into uniformly determined smaller fragments. The DNA ligases permit any two DNA fragments to be joined together. Accordingly, the essence of the recombinant DNA tech- niques is the ability to take a DNA fragment containing any particular gene of interest, say a-interferon, and join it into an appropriate vector sequence, such as a plasmid, to create a hybrid or recombinant DNA molecule. Such a recombinant sequence can be inserted into bacteria, yeast, or mammalian cells and amplify itself by a factor of 30 to 1,000. In this way, many copies of the a-interferon gene can be produced for study. In addition, if the appropriate regulatory machinery is available in the vector, the gene can be expressed, so that large quantities of the corresponding protein are produced for biological study or application. Transformation of Higher Organisms Biologists Can Specifically Insert a Functioning Gene into the Genome of Complex Organisms The revolution in molecular genetics has led us into an understanding of how genes are functionally constructed and has allowed unprecedented access to specific genes and the protein products they encode. Transformation is the process by which DNA molecules that have been isolated (usually by recombi- nant DNA techniques) are introduced into the cell in a way that allows a gene's perpetuation from generation to generation. Much of what we have reamed about genes has been through experimental approaches in transformation that transfer those genes from one place to another-either from cell to cell, organism to organism, or cell to test tube, and then from test tube back into cells and organ- isms. This has allowed us to dissect the DNA of a gene and separate it into specific components; in particular, it has allowed the DNA of a particular gene to be separated into its protein coding information, as specified by the genetic code,

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22 OPPORTUNITIES IN BIOLOGY B~:t ~ ::: :~ :, : Chit EDNA : : it: ~ ~ i: ~d of: Plasmid DNA : ( gnaw ant or Hewn cede : If ~ .: by At: Pl~smid is reed from Serum . _, : : ~ ~ _ mu: ~ DhA in All :nuc us _ : fat DNJ4 is remand ~ ~ from cell news . Ens sir : enzy1n~-are Ivy to cut~open :: the paid and Cut cat a an: frown the DIVA of and: - prism. . ~ ~ ~ _ _ - ::: _ ~ ~ . '6: ~ : : : . _ ~ _ New Me :\ : A me pL2smid ~J - ., . '1 ~: ~ . ~ -8 . OF ~~ Thy m: ends of the pk~mid5 and the =t erg of the new genes are chertaical~ly <'s - " 50 they loll Mach co Ah other-recorr~br7te-to form a new loop conwinir~ the i~t~ gym. This technic ~ Sled "gem splicing" or recombinant DNA technology FIGURE 2-2 Recombinant DNA Technology. [Figure courtesy of Monsanto]

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NEW TECHNOLOGIES AND INSTRUMENTATION 23 and the control elements that participate in the decision about when, where, and under what circumstances a specific gene expresses its encoded information. This decision about when and where a given gene should "play back" its information is of critical importance when one considers that individual plants and animals often contain billions of cells, all of which must act and interact so that the harmony of a healthy, living organism can be preserved. If a gene is expressed in the wrong place, or fails to express in the right place, disastrous problems follow. For this reason, understanding the nature of gene regulation is central to basic research in all aspects of biology. Both the nature of genes and the mechanisms associated with specific dis- eases can be studied effectively by introducing (or transferring) genes into the chromosomes of an individual animal or plant. When transferred to such plant or germ cells of an animal, the new genetic traits are passed on to successive generations, where they reside in every cell of the offspring. Individuals pro- duced by such alterations are called transgenic organisms. Making a Transgenic Animal A Transgenic Animal Is. Produced Initially by a Combination of Microsurgery and Embryological Techniques Much of the experimental work on transgenic animals has been carried out with laboratory mice. Mice have been selected for such experiments because they have the advantages of being small and easy to maintain, having short generations (6 to 8 weeks on the averaged, and producing relatively large numbers of progeny (usually about eight). To produce a transgenic mouse, one first removes fertilized eggs from a female mouse about 12 hours after copulation. After being cultured for a few hours in an incubator, each fertilized egg is injected with a solution of pure DNA through a fine glass capillary needle and monitored through a high-powered microscope. The DNA is injected into one of the two pronuclei, one of which contains the sets of chromosomes originating from the mother and the other from the father. The injected embryos are then reimplanted into Me oviducts of a female mouse and allowed to develop. Typically, if one injects 100 fertilized eggs, about 50 will survive the injection process and perhaps 10 will develop into . . . vlng mice. The mice born are analyzed at 2 to 3 weeks of age to determine which, if any, have incorporated the injected DNA and are therefore transgenic. On average, 2 of every 10 mice born will be transgenic. Several copies of the injected gene will have become integrated into a chromosome of a transgenic mouse, generally in one location, as shown by the Mendelian transmission of the gene to half of its progeny. The transgenic mice that develop from these injected embryos are each potentially the founder of a unique family: Even if the same gene is injected into

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24 OPPORTUNITIES IN BIOLOGY multiple embryos, the different transgenic mice will almost certainly have the gene in a different chromosomal location. Often, the different mice will even show distinct responses to the presence of the transgene. Thus each initial transgenic mouse is used to found a lineage (or family) by mating it to a normal mouse and maintaining its unique genetic properties by selection. Transgenic Mice Have Been Usedfor a Variety of Experiments Transgenic mice have been used to perform detailed analyses of the conse- quences of the presence of particular genes on the organism in which they occur. For example, transgenic mice were produced that earned hybrid genes designed to overproduce human growth hormone. The mice that inherit these growth- hormone genes grow unusually larg~about twice the normal body weight. Among the characteristics associated with large size are lethargy, a shortened life- span, poor reproductive performance of males, and sterility in females. Such complications probably reflect the fact that the size of an organism results from a variety of constraints and compromises, which have been reached in a long evolutionary process. In another line of transgenic mice, human growth hormone is produced under the control of a different regulatory element. The mice of this second line are not sterile, but they are still "big," suggesting that in the long run we may be able to design animals with a desired characteristic but without the accompanying undesirable side effects. Another application using transgenic mice has been the dissection of the regulatory segments of genes expressed in different cell types of the body. In human beings and other mammals, for example, different hemoglobin genes come into play sequentially in embryos, fetuses, and young individuals; the switching mechanisms involved in this sequence have been studied in transgenic mice. The difference in gene action in different parts of the body has also been studied. For example, the spatial control elements of the genes for serum albumin and the digestive enzyme elastase, which specify expression in hepatocytes or pancreatic acinar cells, respectively, are being localized by the application of these methods. The results of such experiments will contribute to our eventual understanding of the mechanisms of tissue-specific control of gene expression. Transgenic mice are also being used in gene therapy experiments that seek to cure genetic abnormalities. In one such example, a mutant strain of mice called "little," which have too little growth hormone, have been "cured" by transferring a growth hormone gene into their chromosomes. The transgenic "little" mice, treated in this way, become nearly normal in size. In similar experiments, a gene encoding a transplantation antigen that had been deleted from a mouse chromo- some was restored to inbred strains of mice. Similarly, mice deficient in gona- dotropin-releasing hormone, which is synthesized in the hypothalamus, remain sexually immature and hence are infertile. This defect has been cured genetically

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NEW TECHNOLOGIES AND INSTRUMENTATION 25 by transferring an intact copy of the gene that encodes the hormone protein into the embryos of mice that are predisposed to develop this inherited abnormality. Such mice become sexually mature and fertile, and therefore "cured." Such experiments can show that specific defects are associated with particu- lar genes. The defects can then be cured by the introduction of normal alleles of these genes. In addition, transgenic mice afford excellent tools for learning about physiology and endocrinology because individuals that Puce particular hormones in unusual quantities can be produced. In such experiments, the genes that are responsible for the production of the hormones can be placed in different regula- tory environments, and they can be active at new sites of synthesis. Examining all of these variables allows a more precise study of mechanisms to be carried out. Creating Transgenic PLants The Creation of Transformed Plants Has Been One of the Most Exciting Developments in Modern BilBY During the past 3 years, systems for the transformation and regeneration of plants have emerged, and the insertion of novel traits has opened a new domain for the study of gene regulation and expression. In addition, the insertion of foreign genes into plants has also provided a powerful tool for modifying plants genetically for applied purposes as well. The successful development of genetic transformation systems in plants has depended on the use of the soil bacterium Agrobacterium tumefaciens, which is capable of infecting plants through wounds to result in crown-gall tumors in many dicotyledonous plants. The tumor-inducing organism carries a large circular plasmid (the Ti plasmid), which contains a piece of DNA (T-DNA) that can be inserted into a plant chromosome. This T-DNA contains genes that encode enzymes for phytohormone synthesis, and the overproduction of these hormones results in tumor formation. Additionally, other genes carried on the Ti plasmid facilitate transfer of the T-DNA. It has been possible to delete most of the genes within the T-DNA, thereby disarming its tumor-inducing genes while preserving its capacity to be transferred. By "filling-in" this modified T-DNA with genes of choice, a powerful vector system for plant transformation has been created. The foreign genes inserted in the T-DNA are often incorporated into the host cell genome; they may subsequently be transmitted in the course of the plant's normal reproduction. The actual transformation system has been greatly simplified so that the process of injection, transformation, and plant regeneration can be carried out easily, producing transformed plants relatively frequently. The system involves the incubation of a genetically modified Agrobacterium that is, one carrying a gene of choice-with either leaf disks or other pieces of tissue capable of forming

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26 OPPORTUNITIES IN BIOLOGY shoots. After the tissue and the bacteria are cultured together for a short time, the bacteria are killed with an antibiotic, and the plant tissue is grown on a medium that allows for the selection and subsequent regeneration of transformed plant cells. Future Prospects The Potentialfor Using Transgenic Organisms to Make Discoveries over the Next 5 or 10 Years Is Vast The techniques of producing ~ansgenic organisms are fast becoming an important part of a wide variety of experimental approaches to questions in biology and medicine. These range from the study of gene regulation, to the development of the immune system, to tests of theories in endocrinology and physiology, to mechanisms of self-tolerance and autoimmune disease, and finally to the study of cancer and other important human diseases.

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NEW TECHNOLOGIES AND INSIRUME=ATION 27 In the long run, it may prove possible to alter the characteristics of farm animals such as pigs, sheep, and cattle, perhaps to provide them with disease resistance or improved physiological responses. This seems a long-range pros- pect at present, and the central focus of research and results will likely remain on transgenic mice for now. In plants, partly because of the ease with which new genotypes can be created in large numbers, the practical applications are apt to come sooner. On the basic side, these techniques have shown that relatively short DNA sequences, only a few hundred nucleotides long, are capable of providing highly specific regulation of gene expression. These results have made possible recent efforts to discover the proteins or nucleic acids that interact with specific DNA regulatory sequences in the gene complex. By such methods, the DNA sequences involved in the regulation of gene expression in specific tissues and developmen- tal processes are being discovered. Other areas for application of these techniques include such processes as embryogenesis and morphogenesis, sexual transmission and inheritance of genes, and the development of new models for the study of diseases. The new technologies in biology are being applied to research problems related to improvements in crop productivity. The commercial development of genetically transformed plants and animals has just begun to emerge as a viable application of these technologies. For example, the development of new transfor- mation techniques for monocotyledonous plants-such as wheat, maize, and rice-coupled with appropriate regeneration technologies will provide results of the greatest economic importance. In medicine the transformation of microbial cells with foreign genes has resulted in the commercial production of such valuable products as insulin, human growth hormone, interferons, and tissue plasminogen activator. MONOCLONAL ANTIBODIES Monoclonal Antibodies Can Be Used as Biological Probes for Specific Molecules Another significant area of biological advance has been the development and application of monoclonal antibodies. Antibody molecules exhibit exquisite specificity for the foreign macromolecular patterns (antigenic determinants gen- erally contained on viruses and bacteria) that initiate their synthesis. Antibodies are synthesized by one class of blood cells, the B lymphocytes, with each B cell having the capacity to synthesize just one type of antibody molecule. The typical immune response to a bacterial antigen is self-limiting and extremely heterogene- ous, both because mature B cells have a short lifetime (a few days) and because the myriad different B cells that are turned on produce many different types of

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28 OPPORTUNE IN BIOLOGY antibody molecules. The development of the monoclonal antibody technique has made it possible to produce virtually unlimited quantities of homogeneous anti- body molecules to antigens of particular interest. The basic idea is to take a transformed (malignant) antibody-producing cell and mutate it so that it can no longer produce its own antibody (Figure 2-3~. Then it is fused to a normal antibody-producing cell to generate a hybrid cell line, having the chromosomes of both parents. Such a cell line, which is potentially immortal, has the capacity to produce unlimited quantities of one particular Me of antibody molecule. Thus, large quantities of homogeneous antibodies of any particular specificity can be produced. An additional powerful tool for obtaining antibodies of particular specif~ci- ties arises from our ability to synthesize peptide fragments of proteins through the use of a peptide synthesizer. By this procedure, a fragment of protein is synthe- sized, coupled to an appropriate carrier protein (a larger molecules, and used to immunize animals. Some fraction of the time, antibodies that can recognize the protein from which the peptide fragment was derived, as well as the peptide fragment itself, are generated. Thus, the antibody response can be directed precisely to a particular region in the protein molecule. In a sense, the peptide antibody approach allows us to fine-tune the specificity of the immune response. Monoclonal antibodies can then be generated from these immune responses. The use of monoclonal antibodies has revolutionized many aspects of funda- mental biology and clinical medicine. It is now possible to obtain monoclonal antibodies for rare and biologically significant or medically interesting molecules. These antibodies can be used to identify and purify these key molecules, and they have enormously facilitated many aspects of the study of development and mo- lecular structure-function relationships. In addition, they have provided countless critical diagnostic reagents [for example, the antibody specific to HIV, the virus associated with acquired immune deficiency syndrome (AIDS)~; in the future, they will be used increasingly as therapeutic reagents. Antibodies have also played a critical role in permitting the genes that synthesize these protein products to be isolated by a blending of recombinant DNA and monoclonal antibody techniques. MICROCHEMICAL TECHNIQUES Microcherrucal Instrumentation Has Had a Powerful Impact on Modern Biology That Is Just Beginning to Be Felt Many of the advances in modern biology are dictated not only by the development of new technologies but also by the development of instrumentation. For example, the instrumentation for the automated synthesis and sequencing of protein and of DNA has been developed over the past 20 years.

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NEW TECHNOLOGIES AND INSTRUMENTATION Mice immunized with antigens in subcutaneous sites Lymph node cells from immunized mouse fused with mouse myeloma cells Antibody-producing hybrid cells (hybridomas) ~( 1 O O O O O ~ 00 C) O ~ O , ~ Hybridomas screened / O O ~ 00~0 0 0 \ for antibody / O ~ .. O 000 0\ production | O O O O O 00 ~ O \ Selected hybridomas are cultured Purified monoclonal antibodies 29 f ~ J L / FIGURE 2-3 Production of monoclonal antibodies. [Adapted from J. F. Keamey, in Fundamental Immunology, W. E. Paul, ed. (Raven, New York, 1984), p. 756]

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30 OPPORTUNITIES IN BIOLOGY Protein Sequencing. In 1967 the first protein sequencer, an automated device for determining the linear order of amino acid subunits in a polypeptide chain starting at one end (the amino terminus) was developed. In the ensuing 20 years, the amount of protein required for sequencing has dropped to 1/10,000 of the earlier amount, to the point that experienced protein chemists can sequence 10 picomoles of protein. The ability to sequence very small quantities of protein is important because even limited amounts of protein sequence data can facilitate cloning of the corresponding gene. Currently a variety of new approaches are being used for even more sensitive detection of amino acids, including fluorescent detection, which should permit sequencing of 10 to 100 femtomoles within the next few years. The importance of this level of sensitivity is that it would permit the direct sequencing of most proteins separated by the most sensitive methodology avail- able-two-dimensional gel electrophoresis. For example, this procedure sepa- rates in one dimension by size and in the second dimension by charge and is capable of separating 1,000 to 5,000 different proteins. When this sensitivity in protein sequencing is attained, many of the genes whose products have been visualized only as spots on a two-dimensional gel can be cloned. Protein Synthesis. A method for protein synthesis has been developed in which the carboxyl terminal amino acid subunit is attached to a resin support and the polypeptide chain is synthesized by a repetitive chemistry that adds one subunit at a time to the growing chain. This approach has been automated, and current state-of-the-art peptide synthesis (and subsequent purification) can pro- duce relatively homogeneous polypeptides as long as 60 residues. Indeed, 140- residue hormones have been synthesized and partially purified. The ability to synthesize peptides is useful in several respects. First, peptide fragments from proteins can be used to generate specific antibodies, which can occasionally bind to the parent protein from which the fragment was derived. Thus peptide synthe- sis can generate useful analytical reagents, or in the clinical realm, diagnostic or therapeutic reagents. Second, peptides can be useful in determining which amino acid residues are key for particular functions (for example, hormones can be synthesized with amino acid modifications at interesting sites). New techniques for protein synthesis, purification, and the joining of peptide fragments will enhance our ability to carry out slTucture-function studies and scale up the synthe- sis of valuable peptides. DNA Sequencing. Two methods are available for DNA sequencing a chemical and an enzymatic method. Both methods generate nested sets of radioactively labeled DNA fragments starting at a single fixed point and terminat- ing at every A, every T. every C, and every G base in four distinct reaction mixtures. These mixtures are run separately on gels that can resolve fragments differing by a single nucleotide, and from the resulting patterns visualized by

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NEW TECHNOLOGIES AND IN=RUME=ATlON 31 radioautography on film, the DNA sequence can be detected. Recently, a fluores- cent chemistry for separately labeling each of the four bases has been developed and the reading of the separated fluorescent-colored fragments automated. This instrument can simultaneously sequence 16 DNA fragments for 300 nucleotides each over an 8-hour cycle time. Accordingly, under ideal conditions 15,000 nucleotides can be sequenced per 24-hour period. In the near future, machines should be developed that are capable of sequencing 150,000 or more nucleotides per day with acceptable error rates. This type of DNA sequencing instrumenta- tion raises interesting questions about the feasibility of sequencing the human genome, a topic that will be discussed elsewhere. DNA Synthesis. A solid-phase method for DNA synthesis has been auto- mated, and a machine is now available that can synthesize hundreds of nucleotide linkages per 24-hour period. Typically, oligomers 30 to 50 nucleotides long are synthesized. Oligonucleotides can readily be joined to synthesize entire genes chemically and those can serve as probes for genes of interest. Oligonucleotide primers can be synthesized rapidly to facilitate DNA sequencing and complemen- tary DNA synthesis. Genes can be mutated readily and specifically by oligonu- cleotide-directed mutagenesis. Finally, in conjunction with the protein sequencer, a powerful strategy is available to clone rare message genes that produce very little RNA and correspondingly small amounts of protein. Once a small amount of amino acid sequence is determined, it can be translated by the genetic code dictionary into a DNA sequence. This sequence can in turn be synthesized as an oligonucleotide fragment that can be used in conjunction with routine recombi- nant DNA techniques to clone the corresponding gene. FLOW CYTOMETRY Flow Cytometry Is Used to Sort Cells A flow cytometer, also known as a fluorescence-activated cell sorter, is an instrument that quantifies fluorescent molecules bound to individual cells or subcellular particles. Many fluorochromes can be used to obtain information about cellular structure and function. Monoclonal antibodies specific for cell surface, cytoplasmic, or nuclear antigenic sites can be coupled to fluorescent molecules such as fluorescein, phycoerythrin, or Texas red. Fluorescent dyes such as propidium iodide and acridine orange can be used to bind to DNA. Because this binding is proportional to the amount of DNA per cell, it reflects the percentage of cells in a population undergoing mitotic division. A number of fluorescent molecules can be used to quantify cell functional activities such as changes in membrane potential, calcium uptake, and intracellular pH. The reper- toire of reagents is growing daily. The flow cytometer is capable of looking at multiple fluorochromes simultaneously. Therefore a combination of monoclonal

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32 OPPORTUNITIES IN BIOLOGY antibodies can be used to label cells, allowing subpopulations of cells that were previously indistinguishable to be identified by means of their unique combina- tion of cell surface markers. The flow cytometer is also capable of sorting populations of cells on the basis of any of the above variables under aseptic conditions that retain cell viability. Cytometric analysis is done by injecting a suspension of cells into a fine stream that passes through a finely focused halogen light source. As a cell passes through the light beam, light-scatter signals are collected by photodetectors. The light-scatter signals give morphological information that can identify some cell subpopulations and separate viable from dead cells. In addition, fluorescence emissions are detected by very sensitive photomultiplier tubes. All signals derived from one cell are digitally processed simultaneously, and the values from that cell are stored by the computer as frequency histograms. The fluorescence intensity can be empirically related to the number of fluorescent molecules bound per cell, which in turn quantitates the number of antigenic sites or dye-binding sites per cell (for example, receptor density or RNA levels). Cell sorting is carried out by oscillating the stream of cells so that droplets are formed, most of which contain only one cell. When the sort begins, cells pass through the light beam. If the cell falls within the defined parameters, an electrostatic charge is placed on the droplet that contains the cell and the charged droplet is deflected into a test tube. Cells can be sorted at rates up to 5,000 cells per second on commercial instruments. Sort rates as high as 100,000 cells per second have been obtained with specially designed cytometers. MICROSCOPY A Revolution in the Application of Light Microscopy Has Occurred Advances in light microscopy have resulted from an integration of the fields of microscope optics, video technology, digital image processing, biology, and chemistry. The remarkable advances in the past few years have been driven by the need for biologists to define the dynamics of the chemical constituents in living cells. Cells perform a variety of cellular functions such as growth, division, movement, intracellular transport, and communication by the coordination of hundreds of complex chemical reactions occurring at distinct times in different regions of cells. Therefore, a complete understanding of the molecular basis for normal, as well as abnormal, cell functions requires methods that can yield both temporal and spatial information about the chemical constituents and chemical reactions in living cells. Modern light microscopy has emerged as a tool that can yield such informa- tion about the chemistry of living cells when used in conjunction with state-of- the-art photodetectors and computers and with specially designed chemical and biological probes. The most important recent advances in optical microscopy

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NEW TECHNOLOGIES AND INSIRUMEN'TATION 33 have been in two major areas: video-enhanced contrast microscopy and low-light- dose microscopy. Video-Enhanced Contrast Microscopy Video-Enhanced Contrast Microscopy Confines the Technologies of Modern Light Microscopy, Video Imaging, and Digital Image Processing Video-er~hanced contrast microscopy results when differential interference microscopy uses a high-resolution video camera instead of photographic fUm to record images. Stray light, which limits contrast, can be suppressed by electroni- cally changing the black and white levels (video offset) and the gain (sensitivity) of the video camera. The resulting improvement in contrast has permitted the camera to detect fine structures that were invisible by conventional light micros- copy to the human eye or film. Biological structures as small as 24 nanometers in diameter or 1/10 the resolving power of a light microscope can be detected in living cells with a time resolution of one video frame (1/30 second). Digital image processing including averaging a few video frames, subtract- ing a background image, and increasing the contrast by computer enhancement methods has improved the image quality even further. This imaging system is capable of recording the movements of biological structures in living cells that had been seen previously only by electron microscopy of killed cells. Video-enhanced contrast microscopy has had an immediate impact on neuro- biology. Nerves consist of a series of elongated cells that propagate electrical impulses and transmit chemical signals between nerve cells and finally to targets such as muscle cells. Many membrane-bound organelles such as synaptic ves- icles, which carry chemical signals to the nerve cell termini, are transported inside the long extensions or axons of these cells. The transport of these organelles is critical for the normal functioning of nerves, but the mechanism for this transport was unknown until recently. The video-enhanced microscope recorded the direc- tions and rates of movement of very small vesicles in squid axons. Low-Light-Dose Microscopy Coupling Biological Cherrustry with Advanced Image Processing Has Permitted Low-Light-Dose Microscopy to Evolve as a Powerful Method of Investigation Low-light-dose microscopy allows the analysis of very weak light signals from luminescent (light-emitting) molecules in or on cells and tissues. Quantitative fluorescence microscopy, when combined with the multitude of biological probes and fluorescent probes now available, offers an approach for gathering complex chemical and molecular information from living cells and tissues. The power of quantitative fluorescence microscopy results from its high sensitivity and specificity, combined with spatial as well as temporal resolution.

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34 OPPORTUNITIES IN BIOLOGY Fluorescence spectroscopic measurements extend the power of fluorescence microscopy to the molecular level. For example, resonance energy transfer allows the distance between two suitably labeled molecules to be determined over the range of 1.0 to 7.0 nm. By the interaction of fluorescent analogues, the assembly of subunits of actin or microtubules can be analyzed in the smallest regions of single cells that can be resolved spatially. Measurement of fluores- cence anisotropy of fluorescent analogues can yield rotary diffusion coefficients, which can be used to determine whether the analogues are free to diffuse or are bound to other structures. Because some fluorescent probes change their spectro- scopic characteristics in response to the chemical environment, changes in the pH, the free calcium ion concentration, or other measures can alter the excitation or the emission properties of environmentally sensitive probes. Measuring the fluorescence at two wavelengths can be used to rapidly quantify the pH, the calcium ion concentration, or other specific variables, allowing temporal and spatial changes in these variables to be determined in living cells. Scanning Acoustic Microscope The Scanning Acoustic Microscope Measures the Elastic Properties of the Cell With the advent of the scanning acoustic microscope as a commercial instru- ment, it became possible to study the elastic properties of cells and biological material on a scale that is similar to that of the optical microscope. The acoustic microscope uses sound waves propagating through liquids rather than optical waves, so that images reflect elastic properties rather than changes in the index of refraction. The relative changes in elastic properties of cells and organelles are often larger than the change in the index of refraction, which means that the contrast may be enhanced in the acoustic images. With living cells attached to substrates, it is easy to monitor the attachment sites, the contour of the cells, and the intercellular networks of fibrils and microtubules. Furthermore, it is possible to monitor the changes in these elastic features in living cells. For the future, acoustic microscopes are being developed that can operate in a cooled liquid such as helium. In such a liquid, the wavelength of sound can be much shorter than that of optical wavelengths. Research instruments have been constructed with a resolution of 200 angstroms, which approaches the resolution of the scanning electron microscope. Scanning Tunneling and Atomic Force Microscope The Scanning Tunneling Microscope Allows One to Image Surfaces with the Resolution of a Few Angstroms The functioning components of the scanning tunneling microscope consists of a sharp tip, usually tungsten, which is mechanically scanned over the surface of the specimen. The scanning tip is prepared in such a way that it consists of a

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NEW TECHNOLOGIES AND INSTRUMENTAT70N 35 single atom. The size of this atom sets the resolving power. The tip is placed within a few angstroms of the surface of the specimen. In this position, the electrons can move, or tunnel, across the barrier between the tip and the specimen, which allows the examination of the density of electrons on the surface being scanned. In the usual case, the electrons are concentrated near the atomic nuclei, and, therefore, the measurement of the electron density gives us a measure of the position of the individual atoms. Since electrons are involved, the specimen used must be conductive. A large amount of work has been done with this new instrument in studying the atomic arrangement on surfaces of single crystals of such materials as silicon and graphite, but the imaging power of the scanning tunneling microscope ex- tends beyond such observations into the realm of biology. Primitive images showing some of the components of a bacteriophage, as well as DNA strands, have been recorded. Such results are more a demonstration of future potential, however, than an informative study. The more useful and definitive work accomplished so far with the scanning tunneling microscope has been with organic molecules. Monomolecular layers of hydrocarbon chains, for example, have been imaged with definition sufficient to resolve the shape and spacing of the periodic array. Molecules of sorbic acid deposited on graphite substrates have been imaged, and their vibrational spectra have been identified. The Atomic Force Microscope Holds Great Promise for Analyzing Biological Specimens Even more informative images of biological molecules may come from the atomic force microscope. In the atomic force microscope, the tip is placed on a cantilever beam, which can deflect as the force on the tip is changed. In practice, the tip is scanned over the specimen, and the variation in force between the atoms on the tip and the atoms in the specimen gives information that is displayed in the image. These forces are small and the deflection of the cantilever minute. Nevertheless, several techniques can be used to measure these small deflections with great accuracy. Because tunneling electrons are not used with the atomic force microscope, it is possible to examine nonconducting samples on the atomic scale. This property indicates the great potential of the instrument for biological studies. MAGNETIC RESONANCE Magnetic Resonance Spectroscopy Is Becoming an Invaluable Toolfor Determining the Structures of Complex Molecules Magnetic resonance continues to grow as an increasingly powerful tech- nique for gaining insights into a wide variety of structural and dynamic aspects of biologically important processes; much of this knowledge is unavailable from any alternative technique. Some of the specific present and future applications in

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36 OPPORTUNITIES IN BIOLOGY elude (1) determination of structures of nucleic acids, proteins, and complex oligosaccharides; (2) measurement of dynamic aspects of these molecules and of interactions between them (for example, interactions between enzymes and sub- strates or between antibodies and antigens); (3) observations of metabolic events with cells, tissues, and isolated organs; and (4) medical applications, including noninvasive high-resolution imaging of humans and observation of metabolic activity in various organs. Knowledge of the three-dimensional structures of proteins and nucleic acids has enormously advanced our understanding of biology; x-ray diffraction of single crystals has contributed greatly. In the past several years advanced tech- niques of magnetic resonance (such as two~imensional spectroscopy) have al- lowed major structural questions to be resolved for molecules in solution (under conditions in which they exist in their native environments). These approaches reveal subtle, but biologically significant, deformations of DNA duplexes and important aspects of protein folding. Insights hake also been gained into struc- tures of complex oligosaccharides that, because they are generally not a single molecular species and do not crystallize, are not acessible to x-ray diffraction techniques. Proteins and nucleic acids interact dynamically to accomplish their specific functions. Static structural techniques, such as x-ray crystallography, reveal little of these dynamic events; in contrast, magnetic resonance provides a powerful tool for studying dynamics: how enzymes bind substrates, how antibodies bind anti- gens, how receptors bind ligands. Metabolic events within cells of all Apes can be directly observed by mag- netic resonance; it is a completely nondisruptive technique for studying the complex interrelations among metabolic pathways, which is not possible with any other approach. These techniques have also been extended to studies of tissues and even intact organs such as the heart. Magnetic resonance can yield three-dimensional images of human patients of the quality of drawings in anatomy texts; it has no requirement for injection of radio-opaque dyes. It is also becoming possible to observe the metabolic state of venous internal organs by extensions of these imaging techniques that allow, for example, noninvasive monitoring of kidney function. Both in areas of basic understandings of the structures and functions of biological molecules and in areas of diagnosis of human disease, magnetic reso- nance technologies offer versatile and powerful methods of gaining crucial knowl- edge inaccessible by alternative approaches. COMPUTERS AND DATA ANALYSIS Computers Are Coming to Play a Central Role in Modern Biology To date, more than 15 million nucleotides and 1 million peptide linkages have been determined for genes and proteins. Computers play a central role in

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NEW TECHNOLOGIES AND INSTRUMENTATION 37 data-base management and pattern recognition. In modem biology it is now possible to take a newly isolated gene (or protein) and search through the entire DNA or protein data banks to determine whether it resembles any known gene (or protein). These so-called homology relationships can provide critical insights into the possible function of the corresponding gene product. Moreover, it is possible to take protein sequences and search for patterns of amino acid subunits that correlate with various forms of secondary structure. With the advent of auto- mated DNA sequencing, the DNA data bank will enlarge rapidly. Hence, there will be a compelling demand for better methods to search for sequence patterns in large data bases. Clearly, these demands will require large and more rapid computers (for example, parallel or concurrent processing) as well as better software for pattern searches. It is important to stress the role large and fast computers will play in deriving the rules of protein folding: that is, how the primary sequence of amino acid subunits directs the three-dimensional folding of the polypeptide chain. Computers will also play a critical role in correlating three-dimensional structures with function. In addition to computer applications in structural biology, the use of expanding ecological data bases will require increasing sophistication as interests in modeling increase. As biology moves toward an ever more detailed analysis of the chemistry of life, computers will play an ever-increasing role in data management, data analy- sis, pattern recognition, and imaging. The Gaining of computer-literate biologists will be essential. Conversely, the training of computer scientists with greater understanding of chemistry and biology presents an immediate and compelling need. BIOLOGY AND THE FUTURE Synergistic Interactions of the New Biology Have Shortened the Time Between Fundamental Observations and Applications The interactions among the various biotechnologies (such as recombinant DNA techniques, monoclonal antibody techniques, and microchemical instru- mentation) are striking. Given a small amount of protein, the corresponding gene can be cloned through the use of protein sequencing, DNA synthesis, and recom- binant DNA techniques. Alternatively, for a gene of known sequence, peptides matching part of the gene product can be produced that can in turn be used to generate antibodies to assay the corresponding gene product. If the gene is unusually short, its entire protein product can be synthesized. Moreover, given the protein sequence of a particular gene product, the corresponding gene can be altered for optimum protein production in bacteria, yeast, or mammalian cells. Such techniques and instrumentation will help stimulate biological research into the next century. Much progress will come from the interaction of types of scientists who are working together for the first time. The new synergistic interactions will present new challenges, however, in areas including methods of

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38 OPPORTUNITIES IN BIOLOGY information analysis and dissemination, funding strategies, training and educa- tion, and methods of balancing single-investigator research with that of large research centers. Our success in addressing these challenges will largely dictate the effectiveness of biology research programs in the United States, and hence the contributions of American biologists to the development of the global data base for the field.