Mauwk 1, (983
see parterl 930304

It has been forty years since I began as a young student in

 

{4 ~ Joshua LEDERBERG

My Own research in what we now call molecular biology, with the
late Francis Ryan. It has been twenty-five years since I fist
began to write extensively about the human importance of molecular
genetic research, the idea that there could be what we would to-
day call a genetic engineering technology. Some time later I felt
that perhaps my correct role and the greatest service as part of
this program was to share with the colleagues who are working to-
day in the laboratory and with a wider audience some of my own re-
flections over this period of time.

Any symposium or lecture on the future has implications of
prophecy. We know that to be a prophet often is a somewhat dan-
gerous profession, because the prophet pretends to foretell the
future and the prophet also tries to help make the future, and in
the very discussion as to which are the possibilities that lie be-
fore us, there must be some effort to guide those choices. We make
the future and we cannot so much foretell it as to try to guide it
and direct it. I think it is of the utmost importance that
there be a wide understanding of the technological possibilities
that are in front of us today to try to afford the most useful di-
rection to these very powerful and very exciting technologies so
that they can be of the most human advantage. But let me first remind
you that this subject has a history; it does not emerge fully
grown like Venus out of the clam shell. It has an origin; it has a
history; there are methods that have been used in genetic engineering
over some period of time. Differentmethods are used for dfferent
purposes,

Let's first have a look at the history of genetics. The sub-
ject can be well said to have begun in ®65 when the monk Gregor Men-
del in Brno did his famous experiments on the crossbreeding of the
garden pea, and for the first time constructed the conception of
independent factors in heredity - what we now call the different
genes. His approach was an abstract one, based entirely on a math-
ematical analysis of the different kinds of progeny that were pro-
duced when plants of different characteristics were cross bred to
One another. He could have had no concept as to the material basis
of these anlage, of what we now call the genes, and for many years
genetics had many productive consequences without an understanding
of the chemical basis of the units of heredity.
2 ~ LEDERBERG

By a curious coincidence, the same year 1865 was essentially
the time at which Friederich Miescher first described a gummy
substance that he extracted from cells of pus that was then asso-
ciated with the nucleus of these cells andwhich he then called the
nucleic acids. The function of that material was quite unknown to
him, and it is quite clear that Mendel knew nothing of Miescher's work
and Miescher knew nothing of Mendel's work and the connection be-
tween these observations would remain obscure for Many years. It
was, in effect, rediscovered only in 1900. Simultaneously three
groups of workers either read his Papers, or did similar experiments
or both, and then to their chagrin had to report that this obscure
worker 35 years earlier had anticipated their results. And that
time is the origin of modern genetic science.

Very promptly after the first reappearance of the concept of the
gene in modern scientific work, a physician, Professor of internal
medicine Archibald E. Garrod observed that the inheritance of
certain metabolic difficulties in the human also followed the laws
of Mendelian segregation and he adduced the ypothesis that in these
cases the way that the gene worked was to affect the production or
the failure of production of specific enzymes in the metabolism of
the organism. This anticipation also laid neglected for very many
years, and I was quite surprised to hear directly from George Beadle
that even at the time that he did his work in 1941 he was unaware
of Garrods findings and concepts, which were pointed out very
shortly thereafter.

Over that leap of time there was the full development of what
is called formal genetics, the description of the patterns 6n in-
heritance from one generation to another, but essentially totally
lacking in any concept as to the chemical basis by which these cha-
racteristics could be transmitted. No one doubted that in the sperm
and in the egg there were chemical substances in which were embodied
the potentialities for development that were associated with the
concept of gene, but as to their actual chemical constitution no-
thing could be said at that time. That association was first made
in an experimental way by Oswald Avery and his colleagues MacLeod
and McCarty. MacCarty lives today as a professor emeritus at the
Rockefeller University in the Rockefeller Institute, and of course
I am very proud of that since that is now the institution over
which I have the privilege to preside.

In 1944, Avery and his group made a determination of the chemi-
3 - LEDERBERG

cal composition of a substance that could be isolated from some
Strains of the bacterial species the pneumococcus that caused pneu-
monia and that this substance, when added to cells of cher strains,
would convert their hereditary characteristics from “type two" to
"type three" or from "type one" to "type two". ‘hese types are
Simply the serological types which are used in the practical iden-
tification through specific antibodies of one form of pneumonia in-
fection from another. But much to Dr. Avery's surprise, and to that
of just about everybody else in the world, this substance, that had
a very interesting biological specificity in being able to convert
the biological behavior of one bacterial form to that of another,
was not a protein, was not an enzyme, but proved to be a kind of
the DNA that Miescher had isolated and described so many years be-
fore.

From 1944 on, .there was little question but that the structure
of DNA and how it functioned in cells might be the central question
in experimental biology. At least so it appeared to me as a young
Student. But there were some difficulties about pursuing that
question further. If one wished to study general biology and
general genetics inAvery's system, you had not only the question of
working with the pneumococcus, which one might think of as being a
rather dangerous organism to deal with for routine experimentation,
also rather difficult to grow inthe laboratory and in other ways
not an easy organism for experimental purposes, but even more serious-
ly at that time there was no concept that bacteria had genes.

They could not be crossed; you could not talk about the segregation

of characteristics in the next hybrid generation. There was no easy
way to correlate one's early findings on the alteration of behavior

in bacteria with the very large body of general biological informa-

tion that we had about other plant and animal species and no way to

connect those findings with the mainstream of genetic analysis.

And So, as a young student very deeply excited by Avery's fin~
dings, it seemed to me that one would wish to do either or both of
two things. The first was tried, and at that time was unsuccessful:
to see if one could use DNA as isolated from cells inthe way that
Avery had done, but to do it with an organism already more familiar
for purposes of genetic investigation. One might have wished to do
it with a mouse or with Drosophila, which were then the best-known
organisms for genetic investigation, but a fungus neurospora was at

hand and seemed like an excellent experimental material for those
4 - LEDERBERG

purposes. This indeed was the organism in which Beadle and Tatum
had just recently developed, for studies infbiochemical genetics,
experimental systems in which one could look for particular kinds of
mutations very much like theones that Garrod had found in the
human, but which in the fungus neurospora could be susceptible to
experimental control in the laboratory in a very convenient way.
For example, some of the mutations that Beadle and Tatum had developed
in neurospora were deficient in the enzymes needed for the production
of certaingpecific growth factors. So that although the original
wild type neurospora organism could grow easily on a simple synthetic
medium and produce its own specific growth factors - its amino acids,
its vitamins, and so on- using only glucose as a carbon source, these
Mutant strains would be defective in their nutrition in various re~
spects, and would therefore grow only if specific nutrient sub-
Stances were added to the medium. this albwed not only for the
definition of very precise examples of genetic change which could
be associated with specific biochemical defects, but also provided
a very powerful experimental tool for looking for new genetic forms
in the cultures of the organism. One needed only to start with a
culture - which might have millions or thousands of millions of cells
in it - that was deficient for growth requiring a specific material,
like the vitamin thiamine or like the amino acid leucine. And if
even one cell were present that had restored to it the normal synthe-
tic capacities of the original organism, that cell would le able to
proliferate, to grow, could be very readily detected by inoculating
large numbers of cells into the simple medium which would permit
the wild type organism to grow, yet which would not permit the mu-
tant organism to grow. Well, that was an elegant and entirely sa-
tisfactory expenmental design, but it didn't word. An I don't
take too many apologies for that particular experiment not working -
it's only in the last year that by the use of rather specialized
methods has it been possible to introduce DNA into these kinds of
fungi in order to accomplish that result.

Well, that was disappointing, but that was only half of the pos-
Sibilities that were presented by the challenge of Avery's discovery.
The other half was to see whether perhaps bacteria had a richer life
than had been attributed to them before, to see whether bacteria
could be crossed. And onee that question was put seriously, I did
go through the literature, I consulted Many experts, and I had to

reach the conclusion that perhaps science had been somewhat hasty
5 - LEDERBERG

in deciding that bacteria were devoid of a sexual process. The only
critical evidence farthis really was no different than that mentioned
by van Leeuwenhoek in the year 1676 in the first discovery of bac-
teria. And when he first saw them under the microscope, he said
these were tiny, globule-like things, that he believed they were
living organisms because they would divide in two and divide in two
again, but he could see no evidenae of any sexual process. But in
contrast to that he had a very graphic description by which protozoa
not only divided in two, but also would mate with one another:

"like dragon flies on the wing" was his attribution. I believe that
it is the very graphic distinction that Leeuwenhoek himself made at
the very foundations of microbiology that led to the deeply-held
myth that bacteria differed from the rest of the living world in
lacking a sexual process. That myth was also engrained in the offi-
cial taxonomy of bacteria in 1875. Ferdinand Cohn put order into
the bacterial world with his publication of the classification of
different forms of bacteria, and he put them all under the heading
of schizomycetes, the fungi that divide only by fission. And so,

of course, who would question the my th when the very name of the
group of organisms was to imply that they had only an asexual method
of reproduction. In fact, however, critical evidence on this

point was: never produced - it was just that no reputable microbiolo-
gist had noticed a sexual process. It would be hard to see under
any circumstances, and there is still great controversy about the
very details of the so called ‘tonjugation" in bacteria at the present
time, and of course since nobody then was really looking for it, it
would have been even more difficult to find.

This was not such an important matter until Avery presented his
challenge that the very crux of the development of molecular biology
would depend on there being a genetics of bacteria, and so this was
what provoked a reinvestigation of that myth, and in just a few
weeks time it was possible to demonstrate that bacteria could be
crossed with one another using an experimental design very similar to
the one I mentioned hefore concerning the efforts to transform neuro-
Spora. One simply took mutants of a bacterium - and here, with the
help of Professor Tatum I used what has become a very famous
organism, Escherichia coli strain K12. Mutants were obtained from
it that possessed the same kind of nutritional defect mentioned
before, and it was really child's play to demonstrate mixing cells

that had different biochemical defects, that they would interact
6 - LEDERBERG

with one another to produce normal type cells. Further studies
left without any doubt that also in bacteria there was a well-
developed genetic system.

This was also the foundation of. all Subsequent experimental
studies using E. coli for this kind of molecular genetic investiga-
tion. Others deserve all of the credit for working out the details
of the chemical foundations of that transfer and even for the direct
demonstration that it is the transmission of DNA from the male cell
to the female cell that is the crucial event in the conjugation
process. I will skip very quickly over Many important contributions
to the further development of our present concepts, and even here I
have to simply summarize very quickly a number of elements.

The relationship between DNA structure and protein structure
received a very important boost by the study of a human genetic
disease - and this should remind you again of Archibald Garrod.

Only in this case the disease was an alteration of hemoglobin asso-
ciated with sickle-cell disease, and it was in 1949 that Linus
Pauling and his colleagues demonstrated that the disease which for
Some years had been known to be caught by an inherited Mendelian
defect was associated with a chemical change in the structure of a
protein, in the structure of hemoglobin. In 1953, also stimulated
by the work of Avery and what had then followed by Hotchkiss and
Chargaff, Watson and Crick worked out the fundamental details of the
physico-chemical structure of DNA, the famous double helix that I
believe nowadays everyone has heard about and possibly many readers
could evey draw by heart. 1953 can be used as a very definite
Starting point for the modern era of DNA investigation since we

were now clearly working with DNA as a chemical entity: not as an
abstraction, not as a mathematical postulate, but a material that
not only could be examined in the test tube as any other material
substance but which could be subjected to the same methods of chemi-
cal analysis as had been developed for polymer chemistry and for
organic chemistry in general. And of course there has been an explo-
Sion of effort since that time. Strangely enough, with all of this
very rapid progress and development in our understanding of DNA and
its biological importance, it was not until 1956 that we had the
correct number for the chromosomes in the human species - another
Scandalous delay over what were the technical possibilities---had
there just been sufficient motivation to look and a sufficiently

critical approach to asking questions of great importante in human
7 ~ LEDERBERG

biology.

By 1960, Arthur Kornberg had reached major milestones in his
program to carry the study of the biochemistry of DNA to the step
of defining the enzyme system which is responsible for that magical
Property by which DNA reproduces itself. Self-reproduction ds of
course the chemical propoerty underlying the very fact that DNA has
genetic properties, that it can transmit information from parents to
offspring and even from one species to another. This no longer seems
SO Magical. We now understand that a DNA molecule acts as a template
on which the newly growing DNA must be assembled correctly in order
to be properly formed. Some of the details of these processes have
been unraveled also thanks to distinguishdd biologists in Italy, no-
tably thanks to important contributions by Arturo Falaschi, Vittorio
Sgaramella and others in Pavia. They played an important role in
the development of the enzymology which has been a necessary part
of the leap from the scientific foundations of dealing with DNA as a
chemical entity and as a substrate for enzymes in 1960 to the first,
you might say, technological accomplishment of splicing together or
joining together two DNA molecules from arbitrary origins in the
test tube and thereby for the first time making DNA molecules under
human design that occur rarely if they do occur at all under natural
conditions.

It was still some years after that that we saw the first actual
application of this knowledge to a problem in medicine. Dr. Y.W.
Kan, on the foundations of the work that Pauling had started, really
got after the DNA sequence - the actual structure of the DNA in
human cells that do and do not have the genetic defect of the sickle
cell hemoglobin - and demonstrated a change in the actual structure
at that point, which is already beginning to be useful for diagnostic
purposes to enable one to detect this disease under prenatal condi-
tions, which had not been possible before.

And, of course, during this decade we have seen the further ex-
plosion of this engineering possibility of putting different kinds
of DNA together at will in a convenient vector, and E. coli K12
continues to dominate the technology as well as the science of this
because so much has been learned about it and because it is a very
convenient organism. The basis of industrial and pharmaceutical
application is not just that one can get the continued replication of
a DNA introduced by design into a bacterial carrier, but also that

under appropriate conditions that DNA can be expressed an the bac-
8 - LEDERBERG

terium and produce the same protein which is its natural

function in the cell from which that DNA was originally obtained.
And from that, one keeps seeing almost every day new reports and new
inventions of uses that can be made of this property of DNA for

practical purposes.