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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
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Representative terms from entire chapter:
transgenic mice
20
OPPORTUNITIES IN BIOLOGY
-
__
~_
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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
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dSlir~ ~ A ~,~,, ;c ~ c~~ _~-
w~ ~_, =~ <~ ~ cc ~
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,
22
OPPORTUNITIES IN BIOLOGY
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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]
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
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
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 Bi°l°BY
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
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.
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
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.
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]
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
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
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
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.
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
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
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
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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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.
