Lecture 1: OVERVIEW AND HISTORICAL BACKGROUND OF MOLECULAR BIOLOGY Flashcards by Hazel Ayala

Vitalism vs Mechanism

Vitalism- intact cells possess a “vital force”; studying life needs looking into intact cells
Mechanism- to study life, cells must be dissected like a machine

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Holism vs Mechanism

Holism- the whole greater than the sum of its parts
Reductionism- all biological phenomena can be reduced into molecules and atoms; obeys the fundamental laws of physics and chemistry

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Two Roots of Molecular Biology

Biochemistry and Microbial Genetics

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Biochemistry vs Microbial Genetics

Biochemistry- structure and properties of biomolecules; “Structural root”
Microbial Genetics- information on the genetic material, its transmission and expression; uses simple systems eg. phages, bacteria; “Informational root”

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The study of the physical and
chemical structure of biological
macromolecules

William T. Astbury (1945)

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Present-day definition:

the study of genes and their
products and how these products
function and interact in the
organization and perpetuation of
living things

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Genetic Material

Identity, Replication, Reading stored information, Regulation of gene expression

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Biomolecules

Synthesis, Degradation, Transport in and out of the cell

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Cellular structure and functions

Supramolecular / ultracellular structures; Functions of specialized cells

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Molecular Basis of Biological Phenomena

Aging, Development, Immune response, Diseases and their treatment

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Approaches to problems: Correlation of structure and function of Collagen (tendon protein)

Triple helix; Additional strength in structural functions

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Approaches to problems: 1. Correlation of structures and functions of DNA

Double stranded
Specific base pairing; For stability and
replicability

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Approaches to problems: 1. Correlation of structures and functions of Cell membrane

Non-polar fatty acid
chains; To prevent free-passing of polar substances

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Approaches to problems: 2. Physical measurements

Ultracentrifugation
Electrophoresis
Infrared Absorption spectroscopy

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Approaches to problems: 3. In vitro approach

Crude and reconstituted extracts

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The logic of Molecular Biology- Arguments are based on:

Efficiency
Development and Evaluation of Models
Strong Inferences

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*During evolution, competition and survival became determinants of efficiency
* Little energy and material are wasted
* eg. Gene expression and regulation

Efficiency

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Models are tentative explanation of how a system works
Tested for validity
Revised/re-designed to fit new experimental evidences

Development and Evaluation of Models

All possible explanations for a particular phenomenon are stated
Possibilities are experimentally eliminated one by one until only one remains – strong inference

Strong Inferences

The Transforming Principle

Frederick Griffith (1928); Transformation experiment on Diplococcus pneumoniae (Pneumococcus)

Explain the Griffith’s experiment

Griffith hypothesized that a “transforming principle” from the heat-killed S strain converted the harmless R strain into the virulent S strain. This experiment laid the groundwork for the discovery that DNA is the genetic material, as later demonstrated by Avery, MacLeod, and McCarty in 1944.

In vitro Transformation

M.H. Dawson (1930) and J.L. Alloway (1932)
Precipitation of the transforming substance
Does not need mice
Heat killed S + live R extract
Contained live S

Explain In vitro Transformation

A transforming principle/agent is responsible for the transformation of the avirulent R type to the virulent S type

Chemical Nature of the Transforming Substance

Ostwald T. Avery, Colin M. MacLeod and Maclin McCarty (1944)

Transforming Substance: General Isolation Steps

1. Bacterial culture (50-75 li) at 37°C for 16 18 hrs 2. Centrifuge 3. Resuspend in 150 ml chilled 0.85 M NaCl 4. Heat kill cells at 65°C and wash with saline solution 5. Saline + 0.5% Na deoxycholate to extract water soluble cell components. 6. Ethanol precipitation 7. Chloroform extraction o remove proteins and lipids 8. Enzyme digestion to remove polysaccharide capsule 9. Ethanol precipitation

Transforming Substance: General Properties

* viscous, silky sheen * loses activity in distilled water * activity retained for months in physiological salt solution * withstands heating at 65°C for 30-60 minutes * loses activity with increased acid concentration

Transforming Substance: Qualitative Chemical Tests

Protein Biuret / Million Test ( – ) Lipid Chloroform/ethanol extraction ( – ) RNA Orcinol (Bial) Weakly ( + ) DNA Dische diphenylamine ( + )

Transforming Substance: Elementary Chemical Test

N/P ratio = 1.67

Transforming Substance: UV Absorption

λmax = 260 nm

Transforming Substance: Enzymatic Analysis protease lipase RNase DNase Heated DNase

protease + transformation lipase + transformation RNase + transformation DNase – transformation Heated DNase + transformation

Transforming Substance: _________ is the transforming principle

DNA

What did Fredrick Miescher contributed and what year?

nuclein; 1869

What is "nuclein"

- The chemical of the nucleus - “a new acidic, phosphorus-containing long molecule” - Isolated from the pus cells in bloody bandage

What are the arguments Arguments against DNA as the genetic material?

- DNA is too simple; proteins are more diverse - DNA is relevant only to some bacteria; protein is more likely the genetic material. - The extract was contaminated / not pure DNA.

Who proposed the Blender Experiment

Alfred Hershey and Martha Chase (1952)

What is the hypothesis in the Blender Experiment?

Hypothesis: * DNA is essential for the production of new phages while protein coats are only for attachment

What is the T2 Phage in the Blender Experiment?

- Lyses cells after 30-60 mins to release new phages * Phages used were labeled * Protein coat with 35S * DNA with 32P

This had clearly that ____ is the genetic material and not ______ ?

The Blender Experiment; DNA; protein

Briefly explain The Blender Experiment?

The Hershey-Chase experiment used bacteriophages (viruses that infect bacteria) to determine whether DNA or protein carries genetic information. They labeled DNA with radioactive phosphorus-32 and proteins with radioactive sulfur-35, as DNA contains phosphorus but no sulfur, and proteins contain sulfur but no phosphorus. After allowing the phages to infect bacterial cells, they separated the viral shells from the bacteria and found that only radioactive phosphorus (DNA) entered the cells and directed the production of new viruses. This demonstrated that DNA, not protein, is the genetic material responsible for heredity.

Structural Studies on the DNA: Pheobus Levene

- DNA is linear - made up of nucleotides with four different bases (ATCG)

Structural Studies on the DNA: Who contributed these? 1 purine : 1 pyrimidine * #A = #T * #C = #G

Erwin Chargaff, Ernst Fischer and R. Hotchkiss

Structural Studies on the DNA: What did Linus Pauling contributed?

DNA is a triple-stranded molecule

Structural Studies on the DNA: What did Maurice Wilkins and Rosalind Franklin contributed?

Provided the X-ray diffraction photograph of crystalline DNA

Who contributed DNA as Double Helix?

James Watson and Francis Crick (1953)