I have come to the end of a very interesting course based on the history of molecular biology. Since molecular biology has been my course of study for the past few years, I really enjoyed the readings from the primary articles written mostly in the 1950's and 60's. This paper was focused on the famous experiments performed by Jacques Monod, Francois Jacob, and Arthur Pardee which showed the first significant observation of the intermediate molecule between DNA and protein. See the bibliography for links to the papers that I found so interesting. Now my bit.
In 1959, molecular biology was firing on all cylinders. There was a great interest in the Western world to describe the phenomena of the molecules of life. One of the epicenters of this fascination was in Paris at the Pasteur Institute. Jacques Monod and Franscois Jacob working in related departments brought their related expertise together, Monod bringing his understanding of the Lac region of the E. coli K12 strain and Jacob bringing his understanding of microbial genetics.
In 1959, molecular biology was firing on all cylinders. There was a great interest in the Western world to describe the phenomena of the molecules of life. One of the epicenters of this fascination was in Paris at the Pasteur Institute. Jacques Monod and Franscois Jacob working in related departments brought their related expertise together, Monod bringing his understanding of the Lac region of the E. coli K12 strain and Jacob bringing his understanding of microbial genetics.
Leading up to 1959, Watson and
Crick had proposed in 1953 the structure of DNA. Following logically from their double helical
structure, the “Central Dogma” was considered and officially enunciated by
Crick in 1958. While the logic of the
age called for DNA to act as a template of RNA synthesis which would in turn
act as template for protein synthesis, the observation of the intermediate RNA
and the nature of this RNA were unknown.
The
Pasteur Institute was well-positioned for making great scientific
discoveries. Before Arthur Pardee took
his sabbatical leave from UC Berkeley to visit the Pasteur Institute, he was
preceded by a number of American post-doctoral fellows who converged on the
Pasteur Institute to hone their skills and their logic in preparation for
illustrious scientific careers. American
visitors to the Pasteur Institute included A.M. Pappenheimer Jr., Martin Polock, Melvin Cohn, Irving Zabin,
Gunther Stent, and others. And Americans
weren’t the only ones visiting the Pasteur Institute. Collaboration was an ideal of the science in
those days allowing Crick to visit from England, and even Eastern Europeans
came to Paris, such as Boris Magasanik.
At the Pasteur Institute an environment of learning by logic and
experimentation had instituted the atmosphere whereby the theories of genetic
regulation could be tried, tested, and improved from their basic heritage up to
nearly what we now know of the phenomena.
There
were a variety of other theories upon which insights were gained as a result of
the PaJaMo experiment. Beadle and
Tatum’s “one gene-one protein” theory had a narrow definition in the years
before the Operon theory. Monod
hypothesized that the induction of proteins was from manipulating preexisting
protein structures rather than nascent protein synthesis that each protein
arose from a different mixture of pre-protein products. The inducer-repressor theories of regulation
were in their infancy. The RNA
intermediate of the central dogma, was at this time no more than a good guess
following the logic of other findings.
In
order to test the resounding theories of the day, Pardee, Jacob, and Monod used
the bacterial conjugation experiments of Jacob, the Beta-galactosidase system
characterized by Monod, and the innovative experimental tweaking of
Pardee.
Jacob had discovered with Elie Wollman in 1955 that Hfr strains of
bacteria are able to attach themselves to F- strains and inject
genetic information. Using this
technique, the PaJaMo experiment crosses Hfr strains and F- strains
of a variety of genotypes for the z and i genes.
Monod had previously described the nature of the products of the z
and i genes. The z gene represents the
commonly mutated region of E. coli DNA that produces mutants that do not create
Beta-galactosidase under any circumstance.
The i gene represents the commonly mutated region in E. coli that causes
constant expression of Beta-galactosidase even in the absence of galactoside
inducers.
Pardee had been studying similar repressible systems at UC
Berkeley and brought a twist on data acquisition in the Beta-galactosidase
system which raised the measurement output from twelve experimental values a
day to something nearer one hundred in the same time. While the he claims the measurements are cruder,
the extra data points allowed for faster experimentation and rapid progress
(Tribute p135).
The most interesting cross that
they performed was an Hfr z+i+ x F- z-i-
of which the zygotes were ultimately z+ z- i+ i-. However, what they found was that for the
first two hours these zygotes acted as z+i- mutants which
constantly express Beta-galactosidase.
Then at two hours, they observed that without the addition of inducer,
the z+ z- i+ i- zygotes would
phenotypically show their genotype and Beta-galactosidase would stop being
synthesized. While inconclusive on the
question of nature of RNA, it is among the first observations of a cytoplasmic
intermediate that arises quickly, allows consistent expression, and degrades
quickly. Repression of the DNA by the
newly synthesized repressor stops the ultimate synthesis of Beta-galactosidase
by means that were not then understood, but were supportive of the logical
assumption of the RNA intermediate.
By the end of the PaJaMo
experiment, the nature of the repressor was of greatest interest. In fact, the article finishes with a direct
question: “What is the chemical nature of the repressor? Should it be considered a primary or a
secondary product of the gene?” Not the
most literary way of finishing a paper, but certainly it emphasized the intense
interest that these scientists had in finding the true nature of the repressor
(Pardee et al. 1959).
By 1961 when Jacob and Monod
published their review of genetic regulatory systems (1961) they were certain
that this repressor must be a primary product of the gene, or in other words
they believed it to be RNA. Then, by
1965 in Monod’s Nobel lecture, he stated emphatically that the Lac i repressor
is a protein. We know today from
molecular biology textbooks that the Lac i repressor is a protein. So what happened to make Jacob and Monod so
sure of their RNA repressor.
Later in the same year as the
PaJaMo experiment, Pardee performed some errant experiments with Louise
Prestidge that should have stopped protein synthesis altogether. Unfortunately, this article was unavailable
on the internet. Since it was published
in 1959, databases do not cover it, and there was no electronic version that I
could find. The main finding however,
was mentioned in Jacob and Monod’s review that we read for class in which they
cited Pardee as eliminating protein as the repressor product of the Lac i gene.
The struggle of finding when the
repressor was definitively characterized as a protein is difficult as
well. Since the characterization took
place before the internet, the laborious task of searching actual paper copies
of the articles is necessary. Only the
most famous of these early articles are captured in digital copies today. For this reason, and the time constraints of
finals, I have not as yet found the article that characterizes the repressor as
a protein.
Realistically, the mistake of an
RNA repressor was small in comparison to the great strides that this experiment
took in describing the regulatory nature of Lac i on the Lac z gene. Students of any introductory molecular
biology course become well versed in the Lac operon theory, the
inducer-repressor theory, and their applicability to the regulation systems in
other organisms. Monod is quoted as saying “Whatever is true for
E. coli is true for an elephant.”(p 181 “Tribute”) While we now know of many instances where
Monod is wrong, we also see the many more ways in which he was right.
Bibliography
Jacob, F., Monod, J. Genetic Regulatory Mechanisms in the Synthesis of Proteins. (1960)
Monod, J. From enzymatic adaptation to allosteric transitions. Nobel Lecture, December 11, 1965.
Pardee, A.
B., Jacob, F., Monod, J. The Genetic Control and Cytoplasmic Expression of “Inducibility”
in the Synthesis of b-Galactosidase by E. coli (1959)
Ullman,
A., Origins of Molecular Biology: A Tribute to Jacques Monod. (2003)
