GEN 272 DNA structure and analysis

GEN 272DNA structure and analysis

Introduction

For a molecule to serve the role of the genetic material – must possess four major characteristics:

replication

Storage of information

Expression of information

Variation by mutation

Genetics have been around for some time – 1800s – figured out the inheritance patterns

Techniques revealing how the inherited characteristics are linked to the underlying processes that cause them have been developed since World War II – molecular basis for inheritance – Molecular Genetics

Replication

Replication of genetic material - mitoses

Fundamental property of all living organisms

Only a part of the genetic potential is expressed at any given point

Serve as a repository of genetic information – expressed or not

Storage of information

Expression of informationComplex process – basis for the concept of information flow within the cell.

Central dogma of molecular genetics – DNA makes RNA which makes proteins

DNA

Transcription

mRNA tRNA rRNA

Ribosome

Translation

Protein

Variation by mutation

Serve as the basis of newly arising variability among organisms - mutation

Mutation – a change in the chemical composition of DNA

A alteration will be reflected during transcription and translation – affecting specific protein

Until 1944, observations favored protein as the genetic material

Proteins and nucleic acid were both considered major candidates for the role of genetic material until the 1940’s

Three factors that contributed to this belief:

Most active research – study of transmission genetics and mutations - proteins as candidate were passively accepted.

Believed structure of DNA to simple for the large amount of chemical variation expected for the genetic material – protein 20 different amino acids.

Protein – abundant in cells and large amount of variety

Some were avirulent (noninfectious) strains – do not cause illness

1927 – Frederic Griffith experimented with several different strains of the bacerium Diplococcus pneumoniae

Difference in virulence – related to the polysaccharide capsule of the bacterium (virulent strains have capsule)

Some were virulent (infectious) strains – cause pneumonia

Evidence favoring DNA as the genetic material

Transformation: Early Studies

Nonencapsulated bacteria are engulfed and destroyed by phagocytic cells in the animal’s cirulatory system – virulent bacteria - not engulfed – multiply and cause pneumonia

Encapsulated bacteria form smooth, shiny-surfaced colonies (S) when grown on an agar culture plate.

Nonencapsulated strains produce rough colonies (R)

This feature allows virulent and avirulent strains to be distinguished easily by standard microbiological culture techniques.

Smooth colony Rough colony

Klug, Cummings & Spencer, 2006

Each strain of Diplococcus – may be one of dozens of different types called serotypes

Serotypes are identified by immunological techniques and are usually designated by Roman numerals.

In the USA types I and II are most common in causing pneumonia.

Griffith used types II and III in his experiment – led to new concepts about the genetic material.

He knew – only living virulent cells would produce pneumonia in mice

If heat-killed virulent bacteria are injected into mice – no pneumonia results

Living avirulant bacteria – no pneumonia results

Griffith’s critical experiment:


Griffith’s critical experiment:

Living IIR (avirulent) cells combined with heat-killed IIIS (virulent) cells

Expected – double injection will not kill mice

Mice died after 5 days

Analysis of blood – revealed a large number of living type IIIS (virulent) bacteria

Regarding the capsular polysaccharide – these IIIS bacteria were identical to the IIIS strain from which the heat-killed cell preparation had been made.

Control mice – injected only with living avirulent IIR bacteria - did not develop pneumonia

This finding ruled out the possibility that the avirulent IIR cells has changed (mutated) to virulent IIIS cells in the absence of heat-killed IIIS

Some type of interaction was required between living IIR and heat-killed IIIS cells

Griffith concluded – heat-killed IIIS bacteria were responsible for converting live avirulent IIR cells into virulent IIIS ones – called this phenomenon transformation

In 1931 – Henry Dawson confirmed observations

Dawson showed that transformation could occur in vitro (in a test tube)

Experimental evidence convincing – chemical substance was responsible for transformation

Cells were centrifuged, collected and heat-killed

Critical question: What molecule serves as the transforming principle?

1944 – Avery, MacLeod & McCarty – reported beyond reasonable doubt – the molecule responsible for transformation was DNA

Began their isolation procedure with large quantities (50 – 75 liters) of liquid cultures of type IIIS virulent cells


Transformation: The Avery, MacLeod, and McCarty experiment

Following homogenization and extraction with the detergent deoxycholate (DOC) – obtain soluble filtrate (still contained the transforming principle)

Protein was removed form the active filtrate by several chloroform extractions – polysaccharides were enzymatically digested and removed.

Precipitation with ethanol – yielded a fibrous mass that still retained the ability to induce transformation of type IIR avirulent cells.

From the original 75-liter sample – the procedure yielded 10 to 25 mg of the “active factor”

Further testing clearly established – transforming principle was DNA

After the publication of their report – Research in transformation intensified

Phage adsorbs to the bacterial cell – some component of the phage enters the bacterial cell

Studies done on the bacterium Escherichia coli and one of its infecting viruses, bacteriophage T2 – confirmed that DNA as the genetic material.

Virus (phage) – consists of a protein coat surrounding a core of DNA


Transformation: The Hershey-Chase experiment

Following infection – the viral information “commandeers” the cellular machinery of the host and directs viral reproduction.

In a short time – many new phages are constructed and the bacterial cell is lysed – releasing the progeny virusses (lytic cycle)

In 1952 – Alfred Hershey & Martha Chase - published results from experiments designed to clarify the events leading to phage reproduction.

Results established the independent function of phage protein and nucleic acid in the reproduction process associated with bacterial cell

Hershey and Chase knew for existing data that:

T2 phages consist of approximately 50% protein and 50% DNA

Infection is initiated by adsorption of the phage by its tail fibers to the bacterial cell.

The production of new viruses occurs within the bacterial cell

DNA or protein (or both) – enters the bacterial cell and directs viral reproduction – which was it?

They used radioisotopes P and S to follow the molecular components of phages during infection.

32 35

DNA contains phosphorus (P) not sulfar - P effectively labels DNA32

Proteins contains sulfar nor phosphorus - S labels protein35

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If E. coli cells are first grown in the presence of P or S – then infected with T2 viruses – the progeny phages will have either a radioactively labeled DNA core or a radioactively labeled protein coat.

3532

Labeled phages – isolated and used to infect unlabeled bacteria

Hershey and Chase demonstrated that most of the P-labeled DNA had been transferred into the bacterial cell following adsorption – while the S-labeled protein remained outside the bacterial cell

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John Spizizen & Dean Fraser independently reported – provided that the cell wall is absent – a virus does not have to be intact for infection to occur.

In 1957 – several reports demonstrated that if E. coli was treated with the enzyme lysozyme – the outer wall of the cell can be removed without destroying the bacterium

Enzymatically treated cells – naked – contain only the cell membrane as their outer boundary – protoplasts (or spheroplast)


Transfection experiments

The outer protein coat structure may be essential to the movement of DNA through the intact cell wall – but it is not essential for infection when spheroplasts are used.

The process of infection by only the viral nucleic acid (transfection) proved that DNA alone contains all the necessary information for production of mature viruses.

What applies to E.coli

applies to E. lephant

GEN 272 DNA structure and analysis

Technology

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