Synthetic DNA

The invention: 



A method for replicating viral deoxyribonucleic

acid (DNA) in a test tube that paved the way for genetic engineering.



The people behind the invention:



Arthur Kornberg (1918- ), an American physician and

biochemist

Robert L. Sinsheimer (1920- ), an American biophysicist

Mehran Goulian (1929- ), a physician and biochemist









The Role of DNA



Until the mid-1940’s, it was believed that proteins were the

carriers of genetic information, the source of heredity. Proteins

appeared to be the only biological molecules that had the complexity

necessary to encode the enormous amount of genetic information

required to reproduce even the simplest organism.

Nevertheless, proteins could not be shown to have genetic properties,

and by 1944, it was demonstrated conclusively that deoxyribonucleic

acid (DNA) was the material that transmitted hereditary

information. It was discovered that DNA isolated from a

strain of infective bacteria that can cause pneumonia was able to

transform a strain of noninfective bacteria into an infective strain;

in addition, the infectivity trait was transmitted to future generations.

Subsequently, it was established that DNA is the genetic material

in virtually all forms of life.

Once DNA was known to be the transmitter of genetic information,

scientists sought to discover how it performs its role. DNA is a

polymeric molecule composed of four different units, called “deoxynucleotides.”

The units consist of a sugar, a phosphate group, and a

base; they differ only in the nature of the base, which is always one of

four related compounds: adenine, guanine, cytosine, or thymine. The

way in which such a polymer could transmit genetic information,

however, was difficult to discern. In 1953, biophysicists James D.Watson

and Francis Crick brilliantly determined the three-dimensional

structure of DNAby analyzing X-ray diffraction photographs of DNA

fibers. From their analysis of the structure of DNA,Watson and Crick

inferredDNA’s mechanism of replication. Their work led to an understanding

of gene function in molecular terms.

Watson and Crick showed that DNA has a very long doublestranded

(duplex) helical structure. DNAhas a duplex structure because

each base forms a link to a specific base on the opposite

strand. The discovery of this complementary pairing of bases provided

a model to explain the two essential functions of a hereditary

molecule: It must preserve the genetic code from one generation to

the next, and it must direct the development of the cell.

Watson and Crick also proposed that DNA is able to serve as a

mold (or template) for its own reproduction because the two strands

ofDNApolymer can separate. Upon separation, each strand acts as a

template for the formation of a new complementary strand. An adenine

base in the existing strand gives rise to cytosine, and so on. In

this manner, a new double-stranded DNAis generated that is identical

to the parent DNA.





DNA in a Test Tube



Watson and Crick’s theory provided a valuable model for the reproduction

of DNA, but it did not explain the biological mechanism

by which the process occurs. The biochemical pathway of DNA reproduction

and the role of the enzymes required for catalyzing the

reproduction process were discovered by Arthur Kornberg and his

coworkers. For his success in achievingDNAsynthesis in a test tube

and for discovering and isolating an enzyme—DNA polymerase—

that catalyzed DNA synthesis, Kornberg won the 1959 Nobel Prize

in Physiology or Medicine.

To achieve DNAreplication in a test tube, Kornberg found that a

small amount of preformed DNA must be present, in addition to

DNApolymerase enzyme and all four of the deoxynucleotides that

occur in DNA. Kornberg discovered that the base composition of

the newly made DNAwas determined solely by the base composition

of the preformed DNA, which had been used as a template in

the test-tube synthesis. This result showed that DNA polymerase

obeys instructions dictated by the template DNA. It is thus said to

be “template-directed.” DNA polymerase was the first templatedirected

enzyme to be discovered.

Although test-tube synthesis was a most significant achievement,

important questions about the precise character of the newly

made DNAwere still unanswered. Methods of analyzing the order,

or sequence, of the bases in DNA were not available, and hence it

could not be shown directly whetherDNAmade in the test tube was

an exact copy of the template of DNA or merely an approximate

copy. In addition, some DNAs prepared by DNA polymerase appeared

to be branched structures. Since chromosomes in living cells

contain long, linear, unbranched strands of DNA, this branching

might have indicated that DNA synthesized in a test tube was not

equivalent to DNA synthesized in the living cell.

Kornberg realized that the best way to demonstrate that newly

synthesizedDNAis an exact copy of the original was to test the new

DNAfor biological activity in a suitable system. Kornberg reasoned

that a demonstration of infectivity in viral DNA produced in a test

tube would prove that polymerase-catalyzed synthesis was virtually

error-free and equivalent to natural, biological synthesis. The

experiment, carried out by Kornberg, Mehran Goulian at Stanford

University, and Robert L. Sinsheimer at the California Institute of

Technology, was a complete success. The viral DNAs produced in a

test tube by the DNA polymerase enzyme, using a viral DNA template,

were fully infective. This synthesis showed that DNA polymerase

could copy not merely a single gene but also an entire chromosome

of a small virus without error.





Consequences











The purification of DNApolymerase and the preparation of biologically

active DNA were major achievements that influenced

biological research on DNA for decades. Kornberg’s methodology

proved to be invaluable in the discovery of other enzymes that synthesize

DNA. These enzymes have been isolated from Escherichia

coli bacteria and fromother bacteria, viruses, and higher organisms.

The test-tube preparation of viral DNA also had significance in

the studies of genes and chromosomes. In the mid-1960’s, it had not

been established that a chromosome contains a continuous strand of

DNA. Kornberg and Sinsheimer’s synthesis of a viral chromosome

proved that it was, indeed, a very long strand of uninterrupted

DNA.

Kornberg and Sinsheimer’s work laid the foundation for subsequent

recombinant DNAresearch and for genetic engineering technology.

This technology promises to revolutionize both medicine

and agriculture. The enhancement of food production and the generation

of new drugs and therapies are only a few of the subsequent

benefits that may be expected.





See also : Artificial hormone; Cloning; Genetic“fingerprinting”;

Genetically engineered insulin; In vitro plant culture;

Synthetic amino acid;Artificial gene synthesis.





Further Reading

Synthetic amino acid

 The invention :



Amethod for synthesizing amino acids by combining
water, hydrogen, methane, and ammonia and exposing the
mixture to an electric spark.



The people behind the invention : 



Stanley Lloyd Miller (1930- ), an American professor of
chemistry

Harold Clayton Urey (1893-1981), an American chemist who
won the 1934 Nobel Prize in Chemistry



Aleksandr Ivanovich Oparin (1894-1980), a Russian biochemist

John Burdon Sanderson Haldane (1892-1964), a British scientist







Prebiological Evolution



The origin of life on Earth has long been a tough problem for scientists

to solve. While most scientists can envision the development

of life through geologic time from simple single-cell bacteria

to complex mammals by the processes of mutation and natural selection,

they have found it difficult to develop a theory to define

how organic materials were first formed and organized into lifeforms.

This stage in the development of life before biologic systems

arose, which is called “chemical evolution,” occurred between

4.5 and 3.5 billion years ago. Although great advances in

genetics and biochemistry have shown the intricate workings of

the cell, relatively little light has been shed on the origins of this intricate

machinery of the cell. Some experiments, however, have

provided important data from which to build a scientific theory of

the origin of life. The first of these experiments was the classic

work of Stanley Lloyd Miller.

Miller worked with Harold Clayton Urey, a Nobel laureate, on the

environments of the early earth. John Burdon Sanderson Haldane, a

British biochemist, had suggested in 1929 that the earth’s early atmosphere

was a reducing one—that it contained no free oxygen. In

1952, Urey published a seminal work in planetology, The Planets, in

which he elaborated on Haldane’s suggestion, and he postulated

that the earth had formed from a cold stellar dust cloud. Urey

thought that the earth’s primordial atmosphere probably contained

elements in the approximate relative abundances found in the solar

system and the universe.

It had been discovered in 1929 that the Sun is approximately 87

percent hydrogen, and by 1935 it was known that hydrogen encompassed

the vast majority (92.8 percent) of atoms in the universe.

Urey reasoned that the earth’s early atmosphere contained mostly

hydrogen, with the oxygen, nitrogen, and carbon atoms chemically

bonded to hydrogen to form water, ammonia, and methane. Most

important, free oxygen could not exist in the presence of such an

abundance of hydrogen.

As early as the mid-1920’s, Aleksandr Ivanovich Oparin, a Russian

biochemist, had argued that the organic compounds necessary

for life had been built up on the early earth by chemical combinations

in a reducing atmosphere. The energy from the Sun would

have been sufficient to drive the reactions to produce life. Haldane

later proposed that the organic compounds would accumulate in

the oceans to produce a “dilute organic soup” and that life might

have arisen by some unknown process from that mixture of organic

compounds.





Primordial Soup in a Bottle



Miller combined the ideas of Oparin and Urey and designed a

simple, but elegant, experiment. He decided to mix the gases presumed

to exist in the early atmosphere (water vapor, hydrogen, ammonia,

and methane) and expose them to an electrical spark to determine

which, if any, organic compounds were formed. To do this,

he constructed a relatively simple system, essentially consisting of

two Pyrex flasks connected by tubing in a roughly circular pattern.

The water and gases in the smaller flask were boiled and the resulting

gas forced through the tubing into a larger flask that contained

tungsten electrodes. As the gases passed the electrodes, an electrical

spark was generated, and from this larger flask the gases and any

other compounds were condensed. The gases were recycled through

the system, whereas the organic compounds were trapped in the

bottom of the system.

Miller was trying to simulate conditions that had prevailed on

the early earth. During the one week of operation, Miller extracted

and analyzed the residue of compounds at the bottom of the system.

The results were truly astounding. He found that numerous organic

compounds had, indeed, been formed in only that one week. As

much as 15 percent of the carbon (originally in the gas methane) had

been combined into organic compounds, and at least 5 percent of

the carbon was incorporated into biochemically important compounds.

The most important compounds produced were some of

the twenty amino acids essential to life on Earth.

The formation of amino acids is significant because they are the

building blocks of proteins. Proteins consist of a specific sequence of

amino acids assembled into a well-defined pattern. Proteins are necessary

for life for two reasons. First, they are important structural

materials used to build the cells of the body. Second, the enzymes

that increase the rate of the multitude of biochemical reactions of life

are also proteins. Miller not only had produced proteins in the laboratory

but also had shown clearly that the precursors of proteins—

the amino acids—were easily formed in a reducing environment

with the appropriate energy.

Perhaps the most important aspect of the experiment was the

ease with which the amino acids were formed. Of all the thousands

of organic compounds that are known to chemists, amino acids

were among those that were formed by this simple experiment. This

strongly implied that one of the first steps in chemical evolution was

not only possible but also highly probable. All that was necessary

for the synthesis of amino acids were the common gases of the solar

system, a reducing environment, and an appropriate energy source,

all of which were present on early Earth.



Consequences



Miller opened an entirely new field of research with his pioneering

experiments. His results showed that much about chemical

evolution could be learned by experimentation in the laboratory.

As a result, Miller and many others soon tried variations on

his original experiment by altering the combination of gases, using

other gases, and trying other types of energy sources. Almost all

the essential amino acids have been produced in these laboratory

experiments.

Miller’s work was based on the presumed composition of the

primordial atmosphere of Earth. The composition of this atmosphere

was calculated on the basis of the abundance of elements

in the universe. If this reasoning is correct, then it is highly likely

that there are many other bodies in the universe that have similar

atmospheres and are near energy sources similar to the Sun.

Moreover, Miller’s experiment strongly suggests that amino acids,

and perhaps life as well, should have formed on other planets.



See also : Artificial hormone; Artificial kidney .



Further Reading :

Synchrocyclotron

The invention: 



A powerful particle accelerator that performed

better than its predecessor, the cyclotron.



The people behind the invention:



Edwin Mattison McMillan (1907-1991), an American physicist

who won the Nobel Prize in Chemistry in 1951

Vladimir Iosifovich Veksler (1907-1966), a Soviet physicist

Ernest Orlando Lawrence (1901-1958), an American physicist

Hans Albrecht Bethe (1906- ), a German American physicist









The First Cyclotron



The synchrocyclotron is a large electromagnetic apparatus designed

to accelerate atomic and subatomic particles at high energies.

Therefore, it falls under the broad class of scientific devices

known as “particle accelerators.” By the early 1920’s, the experimental

work of physicists such as Ernest Rutherford and George

Gamow demanded that an artificial means be developed to generate

streams of atomic and subatomic particles at energies much

greater than those occurring naturally. This requirement led Ernest

Orlando Lawrence to develop the cyclotron, the prototype for most

modern accelerators. The synchrocyclotron was developed in response

to the limitations of the early cyclotron.

In September, 1930, Lawrence announced the basic principles behind

the cyclotron. Ionized—that is, electrically charged—particles

are admitted into the central section of a circular metal drum. Once

inside the drum, the particles are exposed to an electric field alternating

within a constant magnetic field. The combined action of the

electric and magnetic fields accelerates the particles into a circular

path, or orbit. This increases the particles’ energy and orbital radii.

This process continues until the particles reach the desired energy

and velocity and are extracted from the machine for use in experiments

ranging from particle-to-particle collisions to the synthesis of

radioactive elements.

Although Lawrence was interested in the practical applications

of his invention in medicine and biology, the cyclotron also was applied

to a variety of experiments in a subfield of physics called

“high-energy physics.” Among the earliest applications were studies

of the subatomic, or nuclear, structure of matter. The energetic

particles generated by the cyclotron made possible the very type of

experiment that Rutherford and Gamow had attempted earlier.

These experiments, which bombarded lithium targets with streams

of highly energetic accelerated protons, attempted to probe the inner

structure of matter.

Although funding for scientific research on a large scale was

scarce beforeWorldWar II (1939-1945), Lawrence nevertheless conceived

of a 467-centimeter cyclotron that would generate particles

with energies approaching 100 million electronvolts. By the end of

the war, increases in the public and private funding of scientific research

and a demand for higher-energy particles created a situation

in which this plan looked as if it would become reality, were it not

for an inherent limit in the physics of cyclotron operation.





Overcoming the Problem of Mass





In 1937, Hans Albrecht Bethe discovered a severe theoretical limitation

to the energies that could be produced in a cyclotron. Physicist

Albert Einstein’s special theory of relativity had demonstrated

that as any mass particle gains velocity relative to the speed of light,

its mass increases. Bethe showed that this increase in mass would

eventually slow the rotation of each particle. Therefore, as the rotation

of each particle slows and the frequency of the alternating electric

field remains constant, particle velocity will decrease eventually.

This factor set an upper limit on the energies that any cyclotron

could produce.

Edwin Mattison McMillan, a colleague of Lawrence at Berkeley,

proposed a solution to Bethe’s problem in 1945. Simultaneously and

independently, Vladimir Iosifovich Veksler of the Soviet Union proposed

the same solution. They suggested that the frequency of the

alternating electric field be slowed to meet the decreasing rotational

frequencies of the accelerating particles—in essence, “synchroniz-

ing” the electric field with the moving particles. The result was the

synchrocyclotron.

Prior toWorldWar II, Lawrence and his colleagues had obtained

the massive electromagnet for the new 100-million-electronvolt cyclotron.

This 467-centimeter magnet would become the heart of the

new Berkeley synchrocyclotron. After initial tests proved successful,

the Berkeley team decided that it would be reasonable to convert

the cyclotron magnet for use in a new synchrocyclotron. The

apparatus was operational in November of 1946.

These high energies combined with economic factors to make the

synchrocyclotron a major achievement for the Berkeley Radiation

Laboratory. The synchrocyclotron required less voltage to produce

higher energies than the cyclotron because the obstacles cited by

Bethe were virtually nonexistent. In essence, the energies produced

by synchrocyclotrons are limited only by the economics of building

them. These factors led to the planning and construction of other

synchrocyclotrons in the United States and Europe. In 1957, the

Berkeley apparatus was redesigned in order to achieve energies of

720 million electronvolts, at that time the record for cyclotrons of

any kind.





Impact



Previously, scientists had had to rely on natural sources for highly

energetic subatomic and atomic particles with which to experiment.

In the mid-1920’s, the American physicist Robert Andrews Millikan

began his experimental work in cosmic rays, which are one natural

source of energetic particles called “mesons.” Mesons are charged

particles that have a mass more than two hundred times that of the

electron and are therefore of great benefit in high-energy physics experiments.

In February of 1949, McMillan announced the first synthetically

produced mesons using the synchrocyclotron.

McMillan’s theoretical development led not only to the development

of the synchrocyclotron but also to the development of the

electron synchrotron, the proton synchrotron, the microtron, and

the linear accelerator. Both proton and electron synchrotrons have

been used successfully to produce precise beams of muons and pimesons,

or pions (a type of meson).

The increased use of accelerator apparatus ushered in a new era

of physics research, which has become dominated increasingly by

large accelerators and, subsequently, larger teams of scientists and

engineers required to run individual experiments. More sophisticated

machines have generated energies in excess of 2 trillion

electronvolts at the United States’ Fermi National Accelerator Laboratory,

or Fermilab, in Illinois. Part of the huge Tevatron apparatus

at Fermilab, which generates these particles, is a proton synchrotron,

a direct descendant of McMillan and Lawrence’s early

efforts.



See also: Atomic bomb; Cyclotron; Electron microscope;

Field ionmicroscope; Geiger counter; Hydrogen bomb;

Mass spectrograph;Neutrino detector; Scanning tunneling microscope;

Synchrocyclotron



Further Reading :