Research in molecular biology has included chemical, physical,
biological, and theoretical investigations of prebiological conditions
on Earth and, possibly, on other planets; studies of cellular
inclusions; genetic material (DNA and RNA) and coding; as well as energy
transfer in biological systems.
The understanding of prebiological conditions on Earth, and possible
conditions on other planets, depends upon the nature of the complex
chemical species which might be encountered. Scientists have shown that
biologically important compounds, such as amino acids, can be generated
by applying an electrical discharge, ultraviolet radiation, or heat to a
gaseous mixture. Biologically interesting compounds can be removed from
such a system by condensation or absorption; however, in the limited
time and space available in such experiments, many compounds are not
produced in sufficient quantity to be measured.
The National Biomedical Research Foundation (NBRF) and the National
Bureau of Standards (NBS) are conducting an investigation on equilibria
in multielement systems. The distribution of molecular species at
equilibrium is independent of the way equilibrium was reached and is
dependent only on pressure, temperature, and elemental composition. Many
of the conditions which might have arisen naturally can be approximated
by thermodynamic equilibrium. Compounds which can be formed at
equilibrium need no special mechanism to explain their presence.
However, special mechanisms have to be sought for those compounds which
could not be so produced and which would have been required for the
structure and nutrition of the first living organisms.
In the absence of precise knowledge of the composition of the primitive
planetary atmospheres, equilibrium concentrations with a wide range of
temperatures, pressures, and elemental compositions are being
investigated by NBRF and NBS. These investigators have postulated that
the maximum atmospheric pressure may have approached 100 atm if the
primitive Earth was sufficiently hot and if an appreciable portion of
the water on Earth’s surface today was present on primitive Earth. (If
the present oceans were to evaporate, the surface pressure would be
approximately 300 atm.) Low pressures of 10⁻⁶ atm and temperatures
between 500° and 1000° K are being used.
A large range of N, O, C, and H compositions are being investigated for
interesting and plausible combinations of factors. In these calculations
an IBM 7090 computer is being used to obtain data on a very large number
of combinations of chemicals. Other chemical species will be added as
the research continues. Some results of this study give an insight into
the variety of biologically significant chemicals which might have
existed during Earth’s primitive prebiological condition or may now
exist on the surfaces and in the atmospheres of other planets (refs.
[ref.151]-[ref.153]). The general method described by White et al.
([ref.152]), minimizing the free energy of the system, was used. The
solution was approached by an iterative process, starting with an
initial guess of concentrations of the compounds. At each step, _M_+1
linear equations are solved where _M_ is the number of elements in the
system.
In addition to listing of the concentrations of all compounds included
in each problem, the results of three-element problems have been
expressed on a triangular composition diagram for convenience. A coarse
grid of 60 points is used to survey all elemental compositions, with
finer grids being used in regions of particular interest. The calculated
concentrations of the compounds at each composition are stored, and
finally a series of triangular diagrams is printed out, each showing the
concentrations of as many as four compounds at the grid points.
Figure 2 shows the results obtained in the C, H, and O systems. Organic
compounds in concentrations greater than 10⁻²⁰ mole fraction are found
everywhere except where free O₂, is present. Solid carbon theoretically
becomes stable along the lower dashed line at 500° K. However, reactions
producing it are very slow. The supersaturated region beyond the line of
potential carbon formation was also investigated. A threshold was found
where polynuclear aromatic compounds are sufficiently concentrated to
form a liquid phase. These conditions may have been involved in the
primordial formation of asphaltic petroleum.
[Illustration: Figure 2.—_Equilibrium diagram for the system C-H-O._]
Jukes and associates ([ref.154]) at the University of California at
Berkeley have been investigating the code for amino acids in protein
synthesis, the key for translating the sequence of bases in DNA into the
sequence of amino acids in proteins. The amino acid code was solely a
matter of theory until Nirenberg and Matthaei ([ref.155]) at the
National Institutes of Health carried out a crucial experiment. This
experiment bridged the last remaining gap separating theoretical
genetics and test-tube biochemistry. It now became experimentally
possible to search for codes for all 20 amino acids concerned in the
synthesis of proteins.
The amino acid bases of DNA are: A, adenine; C, cytosine; G, guanine; T,
thymine; and U, uracil, which replaces thymine in RNA. There are only 16
ways of arranging A, C, G, and T in pairs. For this and other reasons it
is thought that a triplet of three consecutive bases is needed to code
for each amino acid. The sequences of bases in a strand of DNA are known
to be unrestricted with respect to the order in which they occur;
apparently any one of the four bases can be next to any of the other
four, although, of course, each base must be paired with the
corresponding complementary base in the adjacent strand. Since the same
freedom is true of the amino acid sequences in the polypeptide chains of
proteins, any one of the 20 amino acids can occur next to any other.
Moreover, the sequences in DNA are subject to mutational changes in
which one base replaces another, or bases are added to or deleted from
the DNA. Such rearrangements plus the possibility of lengthening of DNA
molecules are numerous enough to account for all the genetics of living
forms since the first appearance of life on Earth.
Most of our knowledge is based on experiments with synthetic RNA carried
out with extracts of _E. coli_. The majority of the work has been at
Nirenberg’s laboratory at the National Institutes of Health and at
Ochoa’s laboratory at New York University ([ref.155]). Various
combinations of A, C, G, and U were used in preparing the synthetic RNA
molecules that are used in experiments to explore the code. These
molecules are made by incubating a mixture of ribonucleoside
diphosphates with a specific enzyme, polynucleotide phosphorylase. An
important property of this enzyme is that it condenses the nucleoside
diphosphates into polynucleotide strands containing random sequences
depending on the proportion of each base. For example, if the enzyme
were furnished with a mixture of 5 parts of A and 1 part of C, it would
make strands containing, on the average, 25 sequences of AAA, 5 of AAC,
5 of ACA, 5 of CAA, and 1 each of ACC, CAC, and CCA. The proportion of
triplets within the strands of a polynucleotide is reflected in the
proportion of amino acids in polypeptides that are obtained in the
cell-free system. Most of the present knowledge of the amino acid code
is based on this concept. All the proposed codes have been discovered by
this experimental approach where synthetic RNA molecules are used as
"artificial" messenger RNA.
Representative of another class of activities in molecular biology is
the examination of passive ion flux across axon membranes. This work is
being done by Goldman at the National Naval Medical Center. The question
of stimulus transmission by nerve tissue is far from simple, and the ion
concentrations associated with nerve membranes is a significant part of
the answer. Because the space environment may very well produce
alterations in these ion potentials, an investigation of their natures
and significance becomes extremely important. A working theory is now
being developed as a result of this study.
Vital cell processes, chemical transformations, and mechanisms that
provide energy for cell maintenance and activity have been studied by
Kiesow (refs. [ref.157] and [ref.158]) at the Naval Medical Research
Institute. The common objective of all phases of this project is the
elucidation of reaction steps in which energy and matter are transformed
in living systems. Compared with _photo_synthetic organisms,
_chemo_synthetic bacteria offer distinct advantages for the study of
energy assimilation. These studies have led to the following
experimental findings.
With the energy from oxidation of nitrite, NO₂— to nitrate, NO₃— as an
_inorganic_ source, and with added _organic_ chemical energy from the
hydrolysis of adenosinetriphosphate (ATP) to adenosinediphosphate (ADP)
and inorganic phosphate, chemosynthetic bacteria are capable of reducing
diphosphopyridinenucleotide (DPN⁺) to DPNH, in a coupled
oxidoreduction-dephosphorylation. Thus, in the crucial step of
chemosynthesis, _ATP is consumed, not produced_. However, in
simultaneously proceeding cell respiration, the energy donor, DPNH, is
oxidized and generates more ATP than is required for DPN⁺ reduction.
This "breeder cycle" for DPNH—with different ratios of cell respiration
and biosynthesis—results in a net production of either DPNH, or ATP, or
both. Production of DPNH in the cycle leads immediately to the
assimilation of C¹⁴ from HC¹⁴O₃—. These observations explain the
bacteria’s energy source without the classical hypotheses of either
direct phosphorylation or direct CO₂ reduction by inorganic chemical or
electromagnetic energy. The cycle transforms the free energy of nitrite
oxidation into the free energy of the organic compounds. Cell
respiration and elementary biosynthesis proceed through structure-bound
enzyme systems in the same fraction of subcellular particles. Three
components, two cytochromes and one flavoprotein, have been identified.
A thermodynamic analysis of the DPNH "breeder cycle" appears to be
attainable by measurements of redox potentials and calorimetric
determinations of heats of reaction.
Studies are also being conducted by Pollard and associates at
Pennsylvania State University in an attempt to formulate a theoretical
basis for the description of the processes of synthesis, growth,
division, and differentiation of the living cell. Such a theory would be
basic to an understanding of very primitive life forms or prebiological
material which might be found elsewhere in the universe. For these
purposes, studies are being undertaken in macromolecular reproduction
which differ from the studies involving cellular genetic material.
Theories concerning the problem of replication of cellular structures
and information storage in two-dimensional systems are being developed.
Theories are also being developed about the mechanisms which control and
regulate receptor and enzymatic activities within the cell.
One study involved the rate of mutation in cells and disposed of the
suggestion that the process of mutation consists of a "tunneling" of
proton from one base to another in DNA. Such a suggestion can no longer
be advanced as a major explanation of mutations.
Work is also being conducted on the centrifugation of cells of _E.
coli_. It has been shown that cells exposed to as little as 100 g have a
modification in their function. This has been looked at from the point
of view of thymine uptake, which would be concerned with the formation
of DNA, and also from the point of view of the induction of an enzyme,
which would correspond to the transcription of the DNA. Preliminary
experiments in the latter case indicate considerable centrifugation
effect. The thymine uptake is affected, but not nearly as much as
formerly thought. Further work is in progress in this area.
Important work has been completed on the cells of _E. coli_ grown on
maltose, which can be induced to produce betagalactosidase by the
addition of thiomethyl galactoside. If cells are irradiated shortly
after induction, the transcription of the DNA ceases and the enzyme
produced by the messenger RNA is observed to reach a maximum. This
enables the calculation of the half-life of unstable messenger RNA. The
half-life for this decay is readily measurable, and values are given
over a temperature range of 17° C (5.2 minimum) to 45° C (0.56 minimum).
These agree very well with half-lives measured by others by inducing for
short times and measuring the course of enzyme formation. The rate of
transcription is involved in the kinetics of cessation of enzyme
induction, and the rate of transcription can be measured. Arrhenius
plots for this rate and the rate of decay are given, and the activation
energies measured are about 16 000 cal/mole. The cessation of
transcription is linked to the degradation, possibly of only one strand,
of DNA.
Pollard has suggested that one important action of ionizing radiation is
concerned with the transcription of the genetic message into RNA.
Clayton and Adler ([ref.159]) showed that induced catalase synthesis in
_Rhodopseudomonas spheroides_ is inhibited by low doses of X-rays,
giving experimental support to the idea. Pollard and Vogler ([ref.160]),
using cells in which the process of induction involved permease, showed
that there is some sensitivity to gamma radiation. Novelli et al.
([ref.161]) found a reduced sensitivity as compared with colony
formation, but it is still a considerable sensitivity.
The process of induction of an enzyme indicates that the transcription
of the genetic message is repressed by something which can be acted on
by a small molecule, the inducer, to remove repression and permit the
formation of messenger RNA, which then acts to make the enzyme. The
messenger RNA undergoes decay through a process which is still not
clear. Very elegant measurements by Kepes ([ref.162]) show that for the
messenger RNA for betagalactosidase, the half-life is 1.02 min at 37° C
and 2.05 min at 25° C. The time of onset of enzyme formation after
induction was found to be about 3 minutes.
If the process of transcription is indeed sensitive to ionizing
radiation, then the irradiation of cells which have just been induced
should show formation of the enzyme to the extent of formation of new
messenger RNA within a few minutes, plus the formation of the enzyme
while the messenger RNA is decaying. This pattern was found by Clayton
and Adler. The experiments conducted by Pollard and associates amplify
and extend their work and also agree with the work of Kepes ([ref.162]).