2. Genetic material - Bacteriophages Flashcards
What is genetic?
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Characteristics of the genetic material
Contains information (stable form) about cell structure, function, development and reproduction
Accurate replication, so progeny cells have the same genetic information as the parental cell
Capable of change (mutation):
essential for genetic variation, adaptation and evolution
Griffith’s transformation experiment (1928)
first experimental approach to analyse physical nature of heriditary material
Used 2 strains of bacterium of streptococcus pneumoniae -> causes pneumonia
- > S - strain (polysaccharide coat, smooth and highly infectious - virulent) - > R - strain (no polysaccharide coat, rough and harmless - avirulent)
Griffith’s worked with 2 varieties of s - strain:
- > IIS + IIIS
- > differ in chemical composition of polysaccharide coat
- > s - strains can generate R - strains in vitro
- ) Type IIR: living, nonvirulent
- > survives; no bacteria
2.) Bacteria with polysaccharide capsule
Type IIIS: living, virulent
-> Dies; type IIIS: virulent; bacteria
- ) Type IIIS: heat killed, nonvirulent
- > survives, no bacteria
- ) Type IIR: living, nonvirulent + Type IIIS: heat killed, nonvirulent
- > Dies; type IIIS virulent, bacteria
S - type cells can mutate into R - type cells and vice versa
IIS IIR IIS
IIIS IIIR IIIS
Griffith’s transformation experiment - Conclusions
- > S-type cells kills mice - not if heat in-activated first
- > R-type cells cannot kill mice
- > Mixing heat killed S cells with living R cells transforms R cells into virulent S cells
- > A substance - the transforming principle - transferred from S to R cells + changed phenotype
- > Oswald Avery - transforming principle was protein
Avery’s transformation experiments: The search for the “transforming principle”
- ) IIIS bacteria
- ) Lysed with detergent
- ) Centrifuged
- ) Cell extract removed from cell debris
- ) Incubated with living IIR bacteria
- ) Living IIIS cells
- > RNA
- > DNA
- > Protein
- > Polysaccharide
Identity of the “transforming principle”
- ) Mixture of DNA + RNA (from IIIS bacteria)
treat with DNase
2.) Only RNA remains
add RNA to R bacteria (IIR)
Plate on growth medium
3.) No S-transformants, also meaning no RNA
If DNA was added to R bacteria, S transformants are produced and DNA is present.
What is transformation exactly?
A.) Recipient DNA, e.g. IIR bacterium
Double stranded donor DNA, e.g. IIIS DNA
B.) One strand of donor DNA enters cell as the other gets degraded
C.) Formation of triple strand
D.) -> Recombination (strand displacement) by double cross-over
- > Chromosome with segment of heteroduplex DNA - > Replication
=> S. pneumoniae and B. subtilis are amenable to natural transformation
=> Artificial transformation by heat shock or electroporation
Hershey and Chase experiments in bacteriophage T2
Phages can transform bacteria too
Lystic life cycle of phage T2
- ) Attachment of phage to E.Coli and injection of phage chromosome
- > phage chromosome
- > Bacterial (host) chromosome
- > Host E.Coli cell
- ) Breakdown of bacterial chromosome by phage-specific enzyme
-> Bacterial chromosome totally broken down
- ) Replication of phage chromosome, using bacterial materials and phage enzymes
- ) Expression of phage genes produce phage structural components
- > phage heads assembled
- > phage sheath, base plate, tail fibres
- ) Assembly of phage particles
- ) Release of progeny phages by lysis of bacterial wall
The Hershey-Chase experiment (1953)
DNA - contains phosphorus (32P)
Protein - contains sulfur (35S)
- > Radioactive isotopes of sulfur + phosphorus used to label DNA + protein
a. ) Preparation of radioactively labelled T2 bacteriophage
- ) - infect E. Coli + grow in 32P medium -> Progeny phages with 32P labelled DNA
- ) - Infect E. Coli + grow in 35S medium -> Progeny phases with 35S protein
b.) experiment showed DNA to be genetic material of T2
- ) - 32P DNA + blend briefly -> phage ghosts -> Radioactivity recovered in host + passed on to phage progeny
- ) - 35S protein + blend briefly -> Radioactivity recovered in phage ghosts + not passed on to the progeny
Structure of DNA: Key players
Erwin Chargaff (1905 - 2002) Linus Pauling (1901 - 1994) Rosalind Franklin (1920 - 1958) Maurice Wilkins (1916 - 2004) James Watson (1928 - ) Francis Crick (1916 - 2004)
DNA composition
NUCLEOTIDE
phosphate group + pentose sugar (deoxyribose) + nitrogenous base
PURINES (parent compound)
- > 9-membered double ring hydrocarbons
- > Adenine + Guanine
PYRIMIDINES (parent compound)
- > 6-membered single ring hydrocarbons
- > Cytosine + Thymine
Base composition (Chargaff’s rule)
-> 1:1 ratio Purines + Pyrimidines
A + T
C + G
-> %GC varies between organisms
20% Thymine in genome
A = T = 20%
Guanine?
A + T = 40%
%G = 100 - 40 / 2 = 30%
Atomic structure: Franklin + Wilkins
X-ray diffraction analysis
DNA is helical with 2 distinctive regularities of 0.34 nm (one bp) + 3.4 nm (one helical turn)
