Explore Molecular Biology for Neuroscientists at UCL. Navigate the intricate landscape of extrachromosomal replications, plasmids, bacteriophage lambda, and the Polymerase Chain Reaction. Delve into the nuanced use of plasmids, genome allelic variations, and the intersection of bioinformatics with ...
Extrachromosomal Replication
Eukaryotes – Prokaryotes
• Origins of Eukaryotes + evolutionary relationship with Archaea = unknown – 2 models
o Three primary domains (3D) scenario
▪
• Archaea and Eukarya have common ancestor
o Two primary domains (2D) scenario
▪
• Archaea and Bacteria are two primary domains
• Eukarya is a secondary domain that arose from the merging of an archaeon and a
bacterium
Horizontal or Lateral Gene Transfer
• Prokaryotic organisms:
o Can directly transfer DNA without reproduction
o Can pick up DNA from the environment
• Horizontal gene transfer
o Gene transfer that is separate from reproduction – non-sexual movement of genes between
genomes
o 3 main mechanisms
▪ Transformation
• Involves uptake of short fragment of naked DNA by
transformable bacteria
o Classical experiments of Griffith and Avery
▪ Pneumococcal bacteria
• S strain – smooth, pneumonia
• R strain – lab strain, rough, no
pneumonia
▪ Mice injected with:
• S strain = died of pneumonia
• S strain that was heat treated = survived
• R strain that was missing the capsular polysaccharide =
survived
• R strain that was missing the capsular polysaccharide + S
strain that was heat treated = died of pneumonia
▪ Conclusion
,Extrachromosomal Replication
• R-form bacteria transformed into stable S form –
transformation process required head treatment
▪ Conjugation
• Discovered by J. Lederberg and E. Tatum
o 2 strains of bacteria
▪ A: auxotroph for methionine
(met-) and biotin (bio-) +
prototroph for (thr+, leu+,
thi+)
▪ B: auxotroph for threonine
(thr-), leucine (leu-),
thiamine (thi-) + prototroph
(met+, bio+)
o Auxotroph = mutant strain of
microorganism that has nutritional
requirements – cannot grow on
minimal medium – requires certain supplements
o Prototroph = wild type strain of microorganism – grows in minimal medium
– does not require supplements
o Strain A was washed and plated on agar plates made with minimal medium
(MM) – that contains only inorganic salts + carbon source + water
▪ Results
• No colonies
• Backmutation cannot restore prototrophy
o Backmutation = process that causes reversion –
change in nucleotide pair in mutant gene that
restores the original sequence + original phenotype
o Strain B was washed and plated on agar plates made with minimal medium
▪ Results
• No colonies
• Backmutation cannot restore prototrophy
o Strain A and B are mixed for a few hours in medium containing all
supplements before plating on afar made with minimal medium (MM)
▪ Results
• Bacterial colonies at a frequency of 10-7
o Conclusion
▪ Either:
• Some form of recombination of genes occurred between
genomes of two strains → producing prototrophs (wild type
bacteria)
▪ Or:
• One cell leaked substances that the other picked up = cross-
feeding
• Davis – whether physical contact is needed to generate prototrophy – if cross-
feeding is a viable hypothesis
,Extrachromosomal Replication
o U tube
▪ Bacterial strain unable to synthesise certain nutrients is placed in
one arm
▪ Bacterial strain unable to synthesise different required metabolites
is placed in other arm
▪ Medium can be transferred between arms – through pressure or
suction
▪ Bacterial cells cannot pass through centre filter
o After hours of incubation – cells are washed and plated on agar plates with
minimal medium
▪ Results
• No colonies
o Conclusion
▪ Physical contact between two strains is necessary for recombination
• Conjugation needs bacteria to bacteria contact
▪ Transduction
• Involves transfer of DNA from one bacterium into another – via
bacteriophages
• Mechanisms that allow bacteria to exchange genetic information
o Fertility Factor
▪ Transfer of genetic information only occurred in one direction
• One cell acts as donor
o Fertility = ability to donate genetic material
• Other cell acts as recipient
o Cell that lost ability to transfer genetic material
▪ During bacterial gene transfer
• Recipient receives genetic information from donor = changed
▪ Fertility of E. coli
• Could be lost and regained easily
o Fertility factor (F) = ability to donate – a hereditary state
▪ Strains that carry F = can donate genetic information = F+ (male)
• F+ cells do not attach other F+ cells
▪ Strains that lack F = cannot donate = F- (female)
•
o 2 bacteria that are in contact – conjugated
▪ Conjugation by the pilus
• F episome
o Plasmid
• Any extrachromosomal hereditary particle
• Found in all domains of life
• Common in prokaryotes
o Eubacteria (true bacteria)
o Archaea
• Common in eukaryotes (yeasts)
o Eukarya
▪ Episome
• Any form of plasmid that can move in and out of chromosomes
, Extrachromosomal Replication
▪ F episome
• 100kb in length
• Two origins of replication (red)
o oriV – vegetative origin
▪ Used when plasmid is free in the
cell
▪ One copy per bacterial
chromosome
o oriT – transfer origin
▪ Used when plasmid is transferred
into another cell
▪ Cis-acting element located at the beginning of the transfer region
▪ Location where transfer of F plasmid is initiated
▪ Large (33 kb) region with 40 genes
• Transposable sequences (yellow)
o Allow integration of plasmid in different places of the bacterial chromosome
– via homologous recombination with identical sequences
o Transfer of F Episome by conjugation
▪ Tra region
• Most genes in the transfer region are involved in DNA transfer and replication
o At least 12 tra genes are needed for modification and assembly of pilus
molecule + stabilisation of the type 4 secretion system (T4SS)
▪ T4SS – system that allows transfer of DNA from one bacterial cell to
the next
• Contains a number of genes – arranged in
loci named tra and trb
o traA – encodes pilin
o traI – encodes relaxase
o traD – encodes coupling protein
ATPase
o traY – encodes transcription factor – DNA bending
o traM – interacts with DNA and numerous proteins
▪ Pilus
• Hairlike structure 2-3 um long
• Hollow cylinder
• 8 nm diameter with 3 nm axial hole
• F+ cell 2-3 pili
• Pili are involved in all conjugations between
gram-negative bacteria
o Stages
▪ Pilus makes contact recipient cell
▪ Pilus retracts
• Cells come closer together
▪ Cell pairs stabilised
• F plasmid nicked in one strand
o Factors form mating pair formation
and stabilisation
▪ Components of
lipopolysaccharide
▪ OmpA protein
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