Download 1 Early concepts of the gene. Pseudoalleles. Demise of the bead

MAJOR ADVANCES IN UNDERSTANDING EVOLUTION AND HEREDITY
FALL 2015
WEEK 6: OCTOBER 13 AND 15
OCTOBER 13: CONCEPTS OF THE GENE
Early concepts of the gene. Pseudoalleles. Demise of the bead model.
OCTOBER 15: DNA AS THE PRINCIPAL CARRIER OF HEREDITY
Nuclein, Chemical characterization of DNA. Pneumococcal transformation. The double helical
structure of DNA. Semi-conservative replication.
Readings to be Discussed Tuesday October 13
 Clarence Peter Oliver (1940) A reversion to wild-type associated with crossing-over in Drosophila
melanogaster. Proceedings of the National Academy of Sciences (USA) 26: 452-454.
Read the article.
 Melvin M. Green and Kathleen C. Green (1949) Crossing-over between alleles at the lozenge locus in
Drosophila melanogaster. Proceedings of the National Academy of Sciences (USA) 35: 586- 591.
Read the article.
 Seymour Benzer (1955) Fine structure of a genetic region in bacteriophage. Proceedings of the
National Academy of Sciences (USA) 41: 344-354.
Read the article.
Readings to be Discussed Thursday October 15
 Friedrich Miescher 1871. On the chemical composition of pus cells. Hoppe-Seyler’s medizinische
chemische Untersuchungen 4: 441-460. (English translation in: Great Experiments in Biology, Gabriel
and Fogel, eds. 1955. Prentice-Hall: Englewood Cliffs, NJ. pp 233-239).
Read the article.
 Oswald T. Avery, Colin M. MacLeod and Maclyn McCarty 1944. Studies on the chemical nature of
the substance inducing transformation of pneumococcal types: induction of transformation by a
desoxyribonucleic acid fraction isolated from pneumococcus type III. Journal of Experimental
Medicine 79: 137-158.
Read pp 137-138 up to EXPERIMENTAL and pp 152-156.
 Oswald Theodore Avery 1943. Letter from Avery to his brother Roy, Dated May 26, 1943. "The
Professor, the Institute, and DNA" Rockefeller University Press 1976. pp 217-220.
Read the article.
 James Dewey Watson and Francis Harry Compton Crick 1953. Molecular structure of nucleic acids.
Nature (London) 171: 737-738.
Read the article.
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 James Dewey Watson and Francis Harry Compton Crick 1953. Genetical implications of the structure
of deoxyribonucleic acid. Nature (London) 171: 964-967.
Read the article.
 Letter from Francis Crick to Mark Bretcher, May 13, 2003.
 Extract from James Watson, “The Double Helix” 1968
 Matthew Meselson and Franklin William Stahl 1958. The replication of DNA in Escherichia coli.
Proceedings of the National Academy of Sciences (USA) 44: 671-682.
Read the article.
Study Questions
Please hand in Tuesday October 13
1. Why was the production of wild-type in a cross of lzg by lzs unexpected in Oliver's time? What was
the expected result of performing such a cross?
2. What evidence does Oliver cite in arguing that the lozenge wild-type flies obtained in his
experiments are not simply spontaneous reversions of the lozenge mutations?
3. Green and Green conclude on page 590 that there are three separate lozenge genes in tandem (“A
further conclusion…is that the three lozenge loci represent a reduplication of essentially identical
genetic material.”). When tested singly against wild type, each of the three lozenge mutations they
studied is found to be recessive. Believing that each of the three lozenge mutations is in a separate gene,
what explanation do Green and Green offer to account for the observation that flies with two different
lozenge mutations in trans are mutant? What is the correct explanation?
4. In a “Perspectives” article in Genetics (vol 124: 793-796, 1990) “The Foundations of Genetic Fine
Structure”, M.M. Green, referring to the 1949 paper by himself and K.C. Green, writes that the lozenge
locus was “the first to reveal the subdivisibility of the gene”. In view of your answer to question (3)
above, did Green and Green in their 1949 paper really accept the idea of " the subdivisibility of the
gene"? Explain.
5. How did Benzer identify rII mutants for use in his fine-structure studies? What procedure did he
follow in order to assign an rII mutant to one of the two rII cistrons, rIIA or rIIB?
6. What procedure did Benzer follow to obtain a recombination frequency between a pair of rII
mutations?
7. What is the smallest recombination frequency between rII mutations observed by Benzer? Accepting
Benzer's estimates of the map length of phage T4 and accepting the "notions" he cites on page 345, what
would be the approximate number of DNA nucleotides between these two closest rII mutations?
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8. How would you explain the mutants designated by the four horizontal lines under the map in Benzer’s
figure 4?
9. What appears to be Miescher's principal interest in undertaking the purification and characterization
of "nuclein"? What are the main conclusions stated by Miescher? What evidence and arguments does he
present in support of these conclusions?
10. Avery et al. cite the view of Dobzhansky that transformation phenomena are likely to be "cases of
induction of specific mutations by specific treatments". What aspect of the transformation of
pneumonococcal types described by Avery et al. goes far beyond being merely a specific mutagen?
Suggestion: Consider the findings described in the first full paragraph on page 154. Is this all-important
aspect mentioned in the four-point summary at the end of the article?
11. What are the principal biologically relevant structural features of the DNA structure proposed by
Watson and Crick in 1953? What features of the sugar-phosphate backbone and of the base pairs make
it possible to place any base pair at any level of the double helix? Explain.
12. Watson and Crick in their April 1953 paper reject the three-chain model of Pauling and Corey in
which the phosphate groups are on the inside of the molecule and the bases on the outside. W and C
argue that the phosphorus groups in Pauling's model are in the salt form rather than the acid form and
therefore are ionized and negatively charged -- making it likely that they would repel each other too
strongly to achieve a stable structure. An independent argument against any three-chain model could
have been made on the basis of unpublished X-ray diffraction evidence Watson and Crick had from a
research report of Rosalind Franklin, indicating two-fold rotational symmetry perpendicular to the axis
of the DNA chain. Why is such symmetry incompatible with any three-chain model?
13. In the 1958 experiment of Meselson and Stahl, after the number of E. coli cells had just doubled
following transfer of the bacteria from heavy nitrogen growth medium to light nitrogen growth medium,
all the DNA was seen to be of hybrid density. Why is semi-conservative replication, by itself, not a
sufficient explanation of why all the DNA was hybrid at the same time? How do Meselson and Stahl
explain this remarkable result? What about the organization and replication of the DNA in E. coli , not
known at the time, provides the correct explanation?
notes on Miescher:
i) The results of analyses for samples I through IX are given in percent of dry weight, even when the
percent symbol is omitted.
ii) Miescher reports phosphorus as P2O5, only 62/142 or 43.7% of which is phosphorus.
iii) The notation “Pt. =” in Miescher's analyses for nitrogen refers to the method he used for
quantitative gravimetric analysis of nitrogen. It involved heating the sample with soda lime (calcium
oxide and sodium hydroxide) to convert protein nitrogen to ammonia, collection of the ammonia (as a
gas) in hydrochloric acid and precipitation of ammonium chloroplatinate, which was then dried and
weighed.
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iv) The awkward translation at the top of page 239 means that samples V and VIII came from one
preparation and samples VI and IX from a different preparation.
Genes, Alleles and Pseudo-alleles - Some Background
The word “gene” was introduced in 1909 by the Danish geneticist Wilhelm Johannsen.
The term is a shortening of the term “pangene”, introduced by the Dutch botanist Hugo
de Vries to describe the intracellular particles, each specifying a different character, that
he postulated in 1889 to be the determinants of heredity. Rejecting Darwin’s ideas about
the inheritance of acquired characteristics and Darwin’s pangenesis with its gemmules
that pass from somatic cells to the germ cells, de Vries nevertheless named his strictly
intracellular units after Darwin’s pangenesis, obscuring the fact that his ideas about the
physical basis of heredity and variation were entirely different from those of Darwin and
much closer to those of Mendel. For Johannsen, the term “gene” was meant simply to
denote a unit of heredity with no particular structural or mechanistic connotation.
Wilhelm Johannsen
1857-1927
From the time that the chromosome theory became widely accepted until the mid
1950's, genes were generally thought to be like beads on a string. The bead (gene)
could, however, exist in multiple forms, a wild-type form and various mutant forms,
collectively called multiple alleles.
When Oliver and then Green and Green did the work described in this week's
readings, there were two different operational definitions of a gene -- i.e. two different
tests
to determine if two mutations are in the same or in different genes. One test,
Hugo de Vries
1848-1935
called a complementation test or a test for allelism, is to make the double trans
heterozygote, in which one chromosome carries a mutation affecting some character
and the homologous chromosome carries a different mutation affecting the same character. The test
requires that both mutations be recessive to wild-type, as most mutations are. Then, if the mutations are
in different genes, the trans double heterozygote will be wild-type. This is because there will be a wildtype copy of each gene, generally enough to give wild-type or close to wild-type phenotype. If, instead,
both mutations being tested are in the same gene, there will be no wild-type copy and the trans double
heterozygote will therefore be mutant. In that case, the mutations are said to be in the same gene, or that
they are allelic to each other. Thus, considering two mutations a and b, both recessive to wild type, if
the heterozygote a+/+b is mutant, a and b are in the same gene. If, instead, the heterozygote is wildtype, a and b are in separate genes. By this test, all the lozenge mutations studied by Oliver and by Green
and Green are in the same gene because heterozygotes of the form a+/+b are phenotypically lozenge,
rather than wild-type.
A different definition of a gene could be based on the result of crossing two mutations affecting the
same character. If one obtains wild-type recombinants, one might say that the two mutations are in
different genes. On the idea that genes are like beads on a string and that crossing-over happens only in
the strings, a finding of recombinants would indeed mean that the two mutations being tested are in
different genes. Similarly, if no recombinants are found in a sufficiently large-scale cross, it would
imply that the two mutations are in the same gene. By this test, because they recombine, the lozenge
mutations studied by Oliver and by Green and Green would be assigned to different genes.
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Having two different operational definitions for the gene is obviously a recipe for trouble and here was
trouble.
Oliver (1940) offered no definite hypothesis to explain his results, suggesting only that repeats (tandem
duplications) might somehow be involved, possibly via unequal crossing-over. Nine years later, after
serving in the Army in WWII, Oliver's former graduate student Melvin Green, together with his wife
Virginia, then at the University of Missouri, took up the problem. Studying three different lozenge
mutations, Green and Green (1949) proposed that there are three tandemly arranged copies
(“reduplications”) of the lozenge gene and that each of the three lozenge mutations they studied is in a
different one of the three copies. This is described on page 590 of their paper. Their assumption of three
separate lz genes allowed them to retain the bead model, with crossing-over confined to the spaces
between genes. But then, if each lz mutation is in a separate gene, how to explain why each of the lz
double heterozygotes they tested (such as lzi +/+ lzj) is mutant, rather than wild type? After all, on their
hypothesis of a cluster of three lz genes, each trans double heterozygote has wild-type alleles of each of
the three genes. The answer given by Green and Green is “…when a lozenge mutant is present on each
homologous X-chromosome, the wild-type alleles behave as recessive genes (or the lozenge mutants act
as dominant genes…”. In other words, whereas wild-type is definitely dominant to lz in single
heterozygotes (lz/+), in double heterozygotes (lzi +/+ lzj) the wild-type copies of lzi and lzj are somehow
rendered recessive! It is as though a mutation in one gene knocks out the function of an adjacent wildtype gene. This rationale, which came to be known as "position-effect pseudo-allelism" is necessitated
by the false assumption that there is no recombination within genes. But as more and more noncomplementing (allelic) lozenge mutations were investigated, more and more tandem copies of the
lozenge gene had to be imagined in order to maintain the view that there is no crossing-over within
genes -- an obvious absurdity.
Making use of the immense resolving power of the baacteriophage T4 rII system,
being able to detect even one wild-type phage among a hundred million rII mutants,
Semour Benzer in 1955 decisively resolved the pseudoallele problem by
demonstrating that recombination can take place within genes, that is within genes
as defined by complementation tests, as described above. His finding and mapping of
dozens and eventually hundreds of recombining mutations in the rIIA gene unit and
also in the rIIB gene not only signaled the demise of the bead theory of the gene but
Seymour Benzer
also brought the resolution of recombinational analysis into the range of single
1921-2007
nucleotide differences and showed that even at this level the genetic map is linear -like the order of nucleotides in the DNA structure that had been proposed two years earlier by Watson
and Crick. Coming at a time when there was still considerable doubt about the correctness of the WC
model and even about the role of DNA as the physical carrier of hereditary information, Benzer's
finding that recombination can take place within as well as between genes and between mutations whose
distance apart apparently corresponded to only a few nucleotides provided a striking parallel to the
linearity and uniformity of the Watson Crick structure.
The genetic map of phage T4 consists of just one linkage group, corresponding to the single DNA
molecule of the phage. When Benzer wrote his 1955 paper, however, there were some gaps in which no
mutants had yet been isolated and located by crossing with other mutants. This led to the false
impression, mentioned on page 345 of his paper, that there might be three linkage groups (and maybe
three pieces of DNA in the phage head. There are not.).
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Incidentally, although studies of the T4rII region made very important contributions to the
understanding of gene structure, genetic recombination and mutation, the functions of the rII genes are
still unknown!
Notes on Oliver and on Green & Green:
(a) The map locations of the mutations and inversion breakpoints in the inversion chromosomes used by
Oliver are:
y(0.0) Hw(0.1) w(1.5)
dl-49(13)
m(36.1) v(33.0) lz(27.7) lzg lzs
dl-49(41) f(56.7) Bx(59.4)
The numbers in parentheses are map distances in centimorgans on the standard (not inverted) X
chromosome, measured from yellow (y), which is at the end of the euchromatic arm of the X. In the
inversion chromosome, represented above, the segment between the breakpoints, containing m, v and lz,
is inverted relative to the wild-type arrangement, as seen by the fact that the map positions within the
inversion decrease as one goes from the left breakpoint of inversion dl-49 to its right breakpoint.
(b) The map order of the loci in the non-inverted chromosomes used by Green and Green is:
ec(5.5)
ct(20.0) sn(21.0)
lz(27.7)
ras(32.8) v(33.0)
f(56.7)
(c) The paragraph in small type next to Figure 3 on page 350 of Benzer's paper refers to a peculiar
property of the T-even bacteriophages, namely the formation of phage partial heterozygotes. Unless you
are already familiar with phage partial heterozygotes, don't bother trying to understand the paragraph. It
will be explained in class.
Erratum: There is a typographical error in Green and Green at the bottom of page 589. The designation
lz46 + should read + lz46 +
DNA as the Principal Carrier of Heredity - Some Background
Synopsis of previous readings: As we have seen in earlier weeks, the fact that plants and animals are
made up of cells and cell products; that cells originate from pre-existing cells; that the nucleus is the
seat of heredity, that the nuclei of male and female gametes fuse to form the zygote, that this
necessitates a reduction division (meiosis), that it is the chromosomes that are the physical carriers of
heredity; and that the segregation and assortment of hereditary factors discovered by Mendel reflects
the behavior of the chromosomes in meiosis all came to be understood within the space of about threequarters of a century. Recall that the Scottish botanist Robert Brown, in 1833, was the first to apply the
term "nucleus" to the approximately spherical structure regularly seen within cells. [It was also Brown
who described the random displacements of microscopic particles he saw within the vacuoles of pollen
grains, subsequently called "Brownian Motion."] Using the presence of a nucleus as a defining
characteristic of cells, Theodor Schwann and Matthias Schleiden by 1840 had concluded that the cell is
the basic structural unit of all animals and plants but did not recognize that cells arise only from preexisting cells, thinking instead that cells arose from extracellular materials or via condensation around
nuclei. Through the work of several investigators observing cell division in various tissues, in
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developing embryos, and in avian blood cells, notably Robert Remak (1850), it gradually was agreed
that cells arise by division of pre-existing cells, a conclusion made famous by Rudolf Virchow's (1855)
dictum Omnes cellula e cellula -- "all cells from cells". Even before Oskar Hertwig had found that
sperm and egg nuclei fuse during fertilization (1876); before Walther Flemming described the motions
of chromosomes in mitosis (1879); and before Edouard van Beneden discovered that sperm and egg
nuclei contribute the same number of chromosomes (two) to the egg at fertilization
and that all four are present in the nuclei of somatic cells of the horse thread worm
Ascaris (1883) -- the Swiss chemist Friedrich Miescher (1871) reported the
purification and chemical characterization of the phorphorus-rich nuclear material
he called "nuclein" from isolated nuclei derived from human leukocytes. From then
on, studies of the chemistry of inheritance accompanied genetic and cytological
investigations in attempting to understand the physical basis of heredity, eventually
leading to the recognition that DNA is the principal cellular carrier of genetic
information and the determination of its molecular structure.
Friedrich Miescher
1844-1895
Intending to undertake "an attack upon the problem of the chemical constitution of
the cell nucleus", Miescher's initial objective was to obtain a relatively pure preparation of nuclei from
a specific cell type, separated from other cellular material. As starting material, he used fresh surgical
dressings from the university hospital in Tubingen, Germany. After trying several procedures for
obtaining pure nuclei from washed cells in the large quantities needed for the crude chemical analytical
methods of that time, he settled on a protocol involving filtration and washing cells from the dressings
with a sodium sufate solution to obtain what he verified under the microscope to be a relatively pure
preparation of leukocytes, followed by alcohol extraction, proteolytic digestion with crude pepsin and
ether extraction, to obtain a relatively pure preparation of leukocyte nuclei.
Gravimetric analysis of different preparations obtained by this procedure and of a further purified
material he called "soluble nuclein" gave reproducible values for the relative amounts of nitrogen,
phosphorus, and sulfur, leading Miescher to conclude that “…we are not dealing with a fortuitous
mixture, but with a chemical entity or a mixture of very closely related substances, aside from the
presence of small amounts of impurities.” and, because its high phosphorus content set it apart from
proteins that “…we have here a substance sui generis, not comparable with any other group at present
known…” Miescher’s nuclein was mainly DNA, although the presence of sulfur meant that it had not
been completely freed of protein. In subsequent work with nuclein he prepared from salmon sperm,
Miescher obtained more nearly pure DNA, as judged from its relative content of phosphorus, carbon,
and nitrogen and the absence of sulfur. Such material was purified still
further and named "nucleic acid" by Miescher's student Richard
Altmann.
As recounted in the 1910 Nobel lecture of the German biochemist
Albrecht Kossel the phosphorus-containing component of nuclein had
by then been found to contain the four DNA bases (adenine, guanine,
thymine and cytosine), whose covalent structures had been determined
Emil Fisher
by the German chemist Emil Fisher (A and G) and by Kossel (C and T), Albrecht Kossel
(1852-1919)
(1853-1927)
together with phosphoric acid residues and a sugar-like carbohydrate.
The sugar was identified as deoxyribose in 1929 by the Russian-born
biochemist Phoebus Levene working at the Rockefeller Institute in New York, who had studied with
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Kossel in Germany and who also identified the phosphate-base-deoxyribose repeat unit of
DNA. The 3’ – 5’ linkage of the repeat units making up the DNA
polymer chain was worked out during the 1940s by the Scottish chemist
Alexander Todd. The Nobel lectures of Fisher, Kossel and Todd may all
be found at the Nobel web site http://nobelprize.org/.
Phoebus
Levene
The localization of DNA in chromosomes was early evidence that DNA
might be the molecular carrier of heredity. But this idea was eclipsed for about a
decade by general acceptance of the hypothesis suggested by Phoebus Levene in
Alexander Todd
1935, that DNA is simply a polymer of a tetranucleotide sub-unit containing one of
1907-1997
each of the four DNA nucleotides -- a monotonous structure incapable of encoding
the genetic information. This view was reinforced by the widespread view of biochemists that
proteins, with their by then well-known great specificity of action, were more likely to be what genes
are made of, with DNA having only a structural role.
But with the purification of the pneumococcal transforming principle (discovered
by Frederick Griffiths in England in 1928) by Oswald Avery, Colin MacLeod
and Maclyn McCarty at the Rockefeller Institute in 1944 and their demonstration
that it was almost completely pure DNA and that its transforming activity
increased with each step of the purification but was destroyed by DNAse and not
by proteases, and, most important, that the amount of transforming principal
produced by transformed cells is far greater than the amount needed to cause
transformation, it began to be clear that DNA is, in fact, a hereditary material.
Nevertheless, there was no rush to accept the idea. Among the small number of
scientists then concerned with the problem, some argued that Avery’s DNA
preparations might contain small amounts of tightly bound
Oswald Avery
(1877-1955)
protein and that it, not DNA, was the carrier of genetic
information. Nor was it clear that the transformation of
pneumococcal capsular antigen types was not a special case, unrelated to the
general mechanism of heredity.
Upon reading the 1944 paper of Avery et al., Erwin Chargaff, the Austrian-born
biochemist, at Columbia University, was one of the first to realize its great
importance and in that same year turned from his studies of lipids to apply the
newly introduced analytical procedure of paper chromatography to determining the
relative amounts of each DNA base in hydrolyzed samples from various animal,
Erwin Chargaff
plant, and bacterial sources. Chargaff found that the relative amounts of the four
(1905-2002)
bases depart widely from 1:1:1:1 in some species of plants and bacteria, thereby
overthrowing the tetranucleotide hypothesis and making it credible that DNA carries genetic
information. Moreover, although the ratio of A+T to G+C varies between species, Chargaff noted that,
independent of source, A = T and G = C, but did not speculate as to the possible meaning of these
relations. For a lively account by Francis Crick of his realization of the significance of Chargaff's ratios
and for accounts of other developments by Watson, Wilkins and Pauling see the videos at:
http://osulibrary.oregonstate.edu/specialcollections/coll/pauling/dna/video/index.html
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Additional evidence produced in the 1940's and early 1950's for the genetic role of
DNA included: (i) observations by ultraviolet (UV) absorbance microscopy and by
specific staining that the DNA in plants and animals is localized in their
chromosomes; (ii) the observation that somatic nuclei of a given species have the
same DNA content in all diploid cells and that this is twice the amount found in
gametes of the same species; (iii) the finding that the DNA content of nuclei
doubles during interphase, when chromosome number is apparently also doubling;
and the observation that the wavelength of UV light (ca 260 nanometers) most
efficient in making mutations in bacteria is also the wavelength maximum for
adsorption by DNA, whereas the adsorption maximum of proteins is closer to 240
nanometers.
The “phage group” around Max Delbruck at Cal Tech,
Max Delbruck
studying the reproduction of bacterial viruses, became
(1906-1981)
convinced of the hereditary role of DNA when, in 1952,
Alfred Hershey and Martha Chase at Cold Spring Harbor, showed, using
radioactively labeled bacteriophage T2, that the phage injects most of its
phosphorus and considerably less of its sulfur when it infects E. coli and that a
substantial amount of the phosphorus but much less of the sulfur is found in the
progeny phages. Because it was not excluded that a minor but still appreciable
Alfred Hershey (1908-1997)
amount of phage sulfur nevertheless does enter the bacteria, the experiment was
Martha Chase (1927-2003)
not as clean-cut an observation as was the evidence from bacterial
transformation, by then extended to numerous genes and to the bacterium Haemophilus influenzae. But
the Hershey-Chase experiment, coming from a different biological system, one that was then under
intensive study by investigators primarily concerned with the mechanism of gene replication, convinced
members of the phage group that DNA was the thing to concentrate on.
Meanwhile, information about the tertiary (conformational) structure
of DNA was accumulating. At Nottingham in the UK John Gulland
concluded in 1946 from the irreversibility of acid and base titration of
DNA that the structure was held together by a system of hydrogen
bonds that is disrupted by extremes of pH; Various physical
measurements showed the molecules to be long and thin; William
Astbury at the University of Leeds concluded in 1943 from X-ray
William Astbury
diffraction that the rings of the purine and pyrimidine bases are
(1898-1961)
John Masson Gulland
stacked
perpendicular
to
the
long
axis
of
the
molecule,
likening
(1898-1947)
them to “a pile of plates”; and at Kings College, London, in a research report of
December 1952, concluded from X-ray diffraction studies of DNA fibers that the phosphorus atoms are
on the outside of the molecule and that the unit cell of the A form of the molecule is C-centered
monoclinic, implying the existence of two-fold rotational symmetry perpendicular to the fiber axis and,
although not recognized at Kings, that there must therefore be two chains running in opposite directions,
owing to the polarity of the sugar-phosphate single chains, as had been established by chemical studies
of the sugar-phosphate linkages in DNA by Alexander Todd.
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Linus Pauling
(1901-1994)
Also, by 1952, physical chemists, especially Linus Pauling and his colleagues at
CalTech, by means of X-ray diffraction, had determined bond lengths and angles in
a wide variety of molecules, thereby establishing a set of standard bond lengths and
standard bond angles along with rules for the formation of weak bonds such as
hydrogen bonds and for the van der Waals packing distances between various pairs
of atoms--information and rules that would be essential to the model-building effort
that led to the discovery of the DNA structure (and had provided information for
Pauling’s discovery of the protein alpha helix).
By 1951, the stage was set for James Watson and Francis Crick
to begin their ultimately successful attempt, through the molecular model-building
approach successfully developed and used in the discovery of the protein alpha-helix
by Linus Pauling at Cal Tech, to discover the double-helical structure of DNA.
A concise account of the discovery of the structure of DNA may be found in "The
Francis Crick Papers", a web site in the "Profiles of Science" series at the US
National Library of Medicine: http://profiles.nlm.nih.gov/SC/.
James Watson (1928-)
Francis Crick (1916-2004)
Despite the enormous importance of the problem, it appears that Crick
and Watson and, separately, Rosalind Franklin and Maurice Wilkins at Kings College
London were the only ones making a full-time effort to determine the DNA structure.
Pauling's effort at CalTech was rather brief and, unlike the two groups in the UK, he had no
access to the superior X-ray diffraction photographs taken by Franklin. For an account of
Franklin's work see: http://profiles.nlm.nih.gov/KR/Views/Exhibit/narrative/dna.html.
Although Watson and Crick at first worked with a three-chain model as had Pauling,
Crick, early in 1953 concluded from Franklin’s X-ray data that there must be just two
chains, running antiparallel to one another. At the same time, Watson discovered that flat
cardboard models of the DNA bases in their most likely tautomeric forms could form an A-T pair and a
G-C pair. Seeing this, Crick realized that either pair can join to the 1' carbon of deoxyribose in the sugarphosphate chain in a manner that allows any sequence of bases to join the two chains together via
hydrogen bonds in a regular manner consistent with the two-fold symmetry revealed by the X-ray data.
This quickly led to a fairly detailed structure obtained by molecular model building. Thus was
discovered and described in 1953 in two papers in Nature the molecular structure of the principal carrier
of heredity -- the veritable secret of life.
Rosalind Franklin
(1920-1958)
Historical note: In a talk published in 1948, Linus Pauling advanced a compelling idea about how the
gene might replicate. It requires that the gene be made of two mutually complementary parts, each
serving as template for the formation of the other during replication. It is therefore puzzling that in 1952
he and Corey published a proposed structure for DNA with an odd (3) rather than an even number of
chains. Later, Pauling said he was surprised he had not taken account of his own previous idea. As will
be explained in class, the mistake leading to the proposal of a three-chain helix was to not take account
of the contribution of water to the mass of the DNA fibers the unit cell of which Pauling correctly
deduced from Astbury’s X-ray diffraction measurements -- a mistake that apparently caused Watson and
Crick as well to begin their model building with a three-chain structure—also with the phosphates
inside.
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"The detailed mechanism by means of which a gene or a virus molecule produces replicas of
itself is not yet known. In general the use of a gene or virus as a template would lead to the
formation of a molecule not with identical structure but with complementary structure. It might
happen, of course, that a molecule could be at the same time identical with and complementary
to the template on which it is moulded. However, this case seems to me to be too unlikely to be
valid in general, except in the following way. If the structure that serves as a template (the gene
or virus molecule) consists of, say, two parts, which are themselves complementary in structure,
then each of these parts can serve as the mould for the production of a replica of the other part,
and the complex of two complementary parts thus can serve as the mould for the production of
duplicates of itself."
Linus Pauling, from The 21st Sir Jesse Boot Foundation Lecture, Nottingham, UK, 28 May 1948.
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