The Search for the Hereditary Molecule. Scientific Investigations Consider all possible explanations...

The Search for the Hereditary MoleculeThe Search for the

Hereditary Molecule

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Scientific Investigations

Consider all possible explanations

Test explanations with evidence

Scientific explanations are: Predictive Testable tentative

Phases: Rely on logic and crude data in initial probes Identify players with better data Identify the underlying mechanisms

By 1920, the Chromosomal Theory of Inheritance and the chemical composition of chromosomes have been revealed

A. Chromosomal Theory of Inheritance revealed that genes are born on chromosomes

B. Chemical composition of chromosomes: only proteins and nucleic acids, not carbohydrates or lipids

C. therefore the hereditary molecule (genes) must be composed of proteins or nucleic acids

The search is on: false starts point to protein

A.P. Levene - The Tetramer Structure of DNA

Nucleotides

SP

B

Four kinds of DNA nucleotides

The tetramer structure of DNA hypothesis

2. Tetramer structure of DNA hypothesis:

DNA {AdenineGuanineCytosineThymine

chemical analysis to measure the relative abundance of the four nucleotides

mistakenly concluded that the four nucleotides are equally abundant

interpretation

DNA is composed of subunits that possess 1 of each kind of subunit

named the subnunits nucleotide tetramers:

purified nucleotides

Conclusion

A T

GC

DNA has a simple tetramer structure. The structure of DNA is too simple to confer the abilities to reproduce or to store information, so protein must be the hereditary molecule

Levene’s Problems?

• First (poor) techniques for measuring nucleotide composition

• Drew an important conclusion from tenuous data

• Was not suitably tentative about these conclusions

The “Alphabet Analogy”

A reliance on logical argument alone:

1. the hereditary molecule must be capable of coding for vast amounts of information

2. If the hereditary molecule is a protein or NA (both polymers), then it is likely to be composed of subunits that are functionally analogous to letters of an alphabet, with the arrangement of the subunit “letters” spelling out different biological “words” (functions).

3. proteins have 20 subunits (amino acids), DNA only has 4 (nucleotides)

4. this suggested that proteins should be able to store more information ...in fact, it seemed preposterous that all the complexity of life could be spelled out with an alphabet of 4 letters

Problems?

• The investigators underestimated the length of biological “words” (genes)

• Reliance on logic alone hindered demonstration that the explanation was false

The trail is found: circumstantial evidence points to DNA

• Avery et al. (1944)

• Chargaff (1949)

• Hershey and Chase (1952)

Griffiths & Transformation

Griffiths 1928Discovery of Transformation

• Pneumonia bacteria

• Wanted to develop a vaccine

• Had two genetically different strains– Virulent (normal)– Non-virulent (mutant)

• Protocol...


mousevirulent bacteria

injectedmouse died



injectedmouse died

mousenon-vir. bacteria

injectedmouse lived



injectedmouse died


injectedmouse lived

mousedead vir bacteria

injectedmouse lived



injectedmouse died


injectedmouse lived

mousedead vir bacteria

injectedmouse lived

mouselive non-vir. bacdead vir bacteria

mouse died


mouselive non-vir. bacdead vir bacteria

mouse died

virulence genes

Non-virulent bacterium

virulent bacterium

transformation

Avery Avery et alet al, 1944; , 1944; the transformation the transformation bioassay is appliedbioassay is applied

Problems of Interpretation?

a) bacteria are very different from all other life forms, which suggested to researchers then that bacteria might have resulted from a completely different origin of life ...so they might have their own unique hereditary molecule. unwise to generalize to other organisms then

b) circumstantial evidence: the mechanisms by which it worked had not been revealed, only its probable identity

Chargaff, 1949 Levene’s examination of the nucleotide composition of DNA

is repeated with refined methods

1. question: what is the nucleotide composition of DNA like and does it reveal whether DNA could be the hereditary molecule?

2. methods:

a) measured the nucleotide composition of DNA (repeating Levene’s work with more refined methods)

b) for many species, covering the whole spectrum of life (to contend with detractors of Avery’s work who were wary about the limited utility of bacterial studies)

Chargaff, 1949 Levene’s examination of the nucleotide composition of DNA

was repeated with refined chromatographic methods

3. results:

a) each species has its own unique nucleotide composition, as you would expect for the hereditary molecule

b) Chargaff’s Rule: [A] = [T] and [C] = [ G] ...nobody understood why. This suggested that DNA had a structural similarity, and therefore a functional similarity (heredity?) in every species

4. problem of interpretation:

circumstantial evidence: the mechanisms by which it worked had not been revealed, only its probable identity

Hershey & Chase, 1952 - a second bioassay

1. question: is the hereditary molecule protein or DNA?

2. background:

a) viruses inject their hereditary molecule into bacterial host cells, then from inside they take over the cell to reproduce

b) therefore, the viruses themselves separate their hereditary molecule from the rest of their bodies (ghosts)

ghostsghosts

Hershey & Chase, 1952 - a viral bioassay

Protein ***DNA

ProteinDNA ***

virus injects hereditary molecule into host cell

shaker dislodges ghosts from hosts

centrifuge separatesghosts from hosts

virus injects hereditary molecule into host cell

shaker dislodges ghosts from hosts

centrifuge separatesghosts from hosts

radioactivity:***

***

***

ghosts

hosts

ghosts

hosts

Hershey & Chase, 1952 - a viral bioassay

problem of interpretation:

circumstantial evidence: the mechanisms by which it worked had not been revealed, only its probable identity

Watson and Crick, 1953The Structure of DNA

A critical test of two perspectives:

• A long-standing view of life:

V I T A L IS MLife is composed of special processes that

cannot be studied or understood by man

• Understanding life as a mechanistic process:1. Life is the product of evolution2. Evolution is the product of reproduction3. Reproduction is the foundation of life

• Could the structure of the hereditary molecule reveal the mechanistic basis of life?

The hereditary molecule’s structure was expected to reveal:

• The molecular basis of REPRODUCTION

• The molecular basis of MUTATION

• The molecular basis of INFO STORAGE

• The molecular basis by which STORED INFO IS TRNASFORMED TO BIOLOGICAL ACTIVTY

Watson and Crick’s Approach

• They possessed clues about DNA’s 3D shape, dimensions, and nucleotide composition

• They would build hypothetical models, whose dimensions could be compared to the known dimensions of DNA

• They wanted to examine whether a structure whose dimensions fit would explain the molecular basis of the hereditary functions …the big prize

Linus Pauling developed this method when he discoveredthe alpha helix secondary structure of proteins

Electron Microscopy Imaging

• DNA is long and thin

• Diameter = 20 A°

X-ray Diffraction

Clues about 3D shape: several important dimensions

of the molecule

Rosiland Franklin

X-ray Diffraction Image of DNARosiland Franklin

Chargaff’s Rule

Nucleotide composition of DNA:

[A] = [T] and [C] = [G]

Why?

Nucleotides are organized into polynucleotide strands with bases deployed as side chains

3D Molecular Modeling

• Build hypothetical structures

• Measure dimensions

• Compare with known dimensions of DNA

First Consideration:How many colinear strands?

20 A°

too small

20 A°

maybe

20 A°

maybe

20 A°

too big

First considered 3 stranded models

Two stranded models:

• First, tried bases out • Next, tried bases in

didn’t work

Two stranded models with bases facing in:

Measured base pairs:

purine - purine

pyrimidine - pyrimidine

purine - pyrimidine

< 20 A°

> 20 A°

= 20 A°

Possible purine - pyrimidine pairs:

A - C

A - T

G - C

G - T

Chargaff’s Rule

Chargaff’s Rule

Only A-T and G-C pairs form hydrogen bonds(A-C and G-T cannot because their partial charges are not oriented favorably)

C-G A-T

A closer look at the C-G base pair

A closer look at the A-T base pair

The hereditary molecule’s structure was expected to reveal:

• The molecular basis of REPRODUCTION

• The molecular basis of MUTATION

• The molecular basis of INFO STORAGE

• The molecular basis by which STORED INFO IS TRNASFORMED TO BIOLOGICAL ACTIVTY

The molecular basis of REPRODUCTION

DNA is double stranded

The two strands can UNZIP

Single strands can act as TEMPLATES to order the synthesis of a second strand

Addition of only COMPLEMENTARY nucleotides to a new elongating strand ensures that sequence information is

preserved in new strands

REPLICATION

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The molecular basis of MUTATION

Accidental addition of non-complementary nucleotides to a new elongating strand

or

Chemical modification of a nucleotide, rendering it non-complementary

The molecular basis of INFORMATION STORAGE

Neighboring nucleotides with a nucleotide pair are complementary (contributing to ability to reproduce)

Neighboring nucleotides within a polynucleotide strand suffer no such limitation - any nucleotide can occupy such a site

Information is stored as specific nucleotide sequences within polynucleotide strands (or sequences of nucleotide pairs)

The molecular basis of

TRANSFORMATION OF STORED INFORMATION TO

BIOLOGICAL ACTIVITY

Required elucidation of

transcription, translation, and the genetic code

…which about 15 years

The Beginning

The Search for the Hereditary Molecule. Scientific Investigations Consider all possible explanations...

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