NEUR0006
MOLECULAR BIOLOGY FOR
NEUROSCIENTISTS
,EXTRACHROMOSOMAL REPLICATION
Origin of eukaryotes and their evolutionary relationship with Archaea is a major biological
question
- Argument about scenarios began in 1977
Two scenarios:
I. Classical view-three primary domains
a. Archaea and Eukarya have common ancestor
II. Two primary domains
a. Archaea and Bacteria are two primary domains, whereas Eukarya is a
secondary domain that arose from the merging of archaeon and a bacterium
PLASMIDS
Plasmids fall into two groups:
I. Conjugative
a. Promote sexual conjugation between bacterial cells resulting in a conjugative
plasmid spreading from one cell to all other cells in a bacterial culture
b. Conjugation and plasmid transfer controlled by tra genes
i. Present on conjugative plasmids but absence on non-conjugative types
II. Non-conjugative
a. Do not promote sexual conjugation
Plasmid Classification
I. Fertility/F plasmids
Carry only tra genes and have no characteristic beyond the ability to promote
conjugal transfer of plasmids
II. Resistance/R plasmids
Carry genes conferring on the host bacterium resistance to one or more
antibacterial agents
HORIZONTAL/LATERAL GENE TRANSFER
Vertical: transfer between parent and offspring normally generating new cells = produces
the next generation
Horizontal: prokaryotic organism directly transfers DNA without reproduction or pick up DNA
from the environment
Movement of genetic material between unicellular and/or multicellular organisms by
means other than reproduction
I. Bacterial Transformation
II. Bacterial Transduction
III. Bacterial Conjugation
HORIZONTAL GENE TRANSFER VIA TRANSFORMATION
a. Uptake of short fragments of naked DNA by transformable bacteria
b. 10 genes can be picked up
c. Long fragments are unstable because cell membrane has to open
, d. Bacterial transformation provided first support that NDA is genetic material in
classical experiemnts by Griffith and Avery
Conclusion: R-form bacterial transformed into a stable S form and the
transformation process required some factor form the heat killed S sample
Factor originally believed to be a protein because DNA was viewed
as a monotonous molecule of uniformity that would have no
biological specificity
In the 40s, it was revealed that the DNA was the demonstrating
factor
o Watson and Crick solidified the concept that DNA was the
genetic material
HORIZONTAL GENE TRANSFER VIA TRANSDUCTION
Transfer of DNA from one bacterium into another via bacteriophages
Bacteriophage: viruses that specifically infect bacteria
I. Generalised transduction: transfer of any gene from one bacterium to another at
low frequency
1. When a bacterial cell is infected with a phage, the lytic cycle may occur
2. During lytic infection, the enzymes responsible for packaging viral DNA into the
bacteriophage packages the host DNA accidently
= produces a transducing particle which cannot lead to viral infection because
they do not have viral DNA
, 3. On the lysis of a cell, transducing particles released along with normal virions
4. Lysate contains a mixture of normal virions and transducing particles
5. When lysate infect cells, small proportion receives transducing particles that inject the
DNA they packaged from a previous host bacterium
a. DNA cannot replicate but can recombine with the DNA of the new host
II. Specialised transduction: efficient, selective transfer of small region of bacterial
chromosome
HORIZONTAL GENE TRANSFER VIA CONJUGATION
Conjugation: mechanism of genetic transfer involving cell-to-cell contact
- Plasmid encoded mechanism
, - Conjugative plasmids use the mechanism to transfer copies of themselves to new host
cells
- Involves a donor cell and a recipient cell
- Genetic elements that cannot transfer themselves can become immobilised during
conjugation
Discovered by J. Lederberg and E. Tatum in 1946
Strain A: auxotroph for methionine (met) and biotin (bio) and
prototroph for (thr+, leu+, thi+)
Strain B: auxotroph for threonine (thr), leucine (leu-), thiamine
(thi-) and prototroph (met+, bio+)
Strain A washed and plated on agar plates made
with minimal medium (MM) that contains only
inorganic salts, a carbon source, and water
No colonies
Growth on the agar plate does not enable
backmutation to restore prototrophy for
methionine and biotin
Strain B was washed and plated on agar plates
made with minimal medium (MM)
No colonies
Backmutation cannot restore prototrophy
Strain A and B mixed in medium containing all
supplements before plating on agar with MM
Colonies at a frequency of 10-7
Suggests that recombination of genes taken place
between the genomes of the two strains to produce
the prototrophs or one cell leaked substances that
the other picked up (cross-feeding)
Is physical contact required to generate
prototroph?
- Suspension of bacterial strain unable to
synthesise certain nutrients is placed in one
arm of U-tube
- A strain genetically unable to synthesise
required metabolites is placed in the other arm
- Liquid may be transferred between the arms by
applying pressure or suction, but bacterial cells
cannot pass through
- After several hours of incubation, cells were
washed and plated on agar plated made with
MM
o No colonies were formed
o Requires direct contact for conjugation
and recombination
FERTILITY FACTOR (aka F
plasmid)
Allows genes to be transferred from one bacterium carrying the factor to another bacterium
lacking the factor by conjugation
, - Recognised that transfer only
occurred in one direction
o One cell act as a donor
o Lost its ability to transfer
genetic material
o Other cell acts as a recipient
- During bacterial gene transfer, the
recipient receives genetic information
from the donor and is thereby changed
- Fertility (ability to donate) of E.coli could be regained and lost
o Suggests that the donor ability itself is a herefitary state imposed by a fertility
factor (F)
- Strains that carry F can donate and are designated F+ (male)
- Strains that lack F cannot donate and are recipients are designated F- (female)
- F+ cells do not attach to other F+ cells
F EPISOME
Episomes are special form of plasmid that can also
traffic in and out of chromosomes
- ~100kb in length
- Two origins of replication:
o oriV: used when the plasmid is free in the
cell (one copy per bacterial chromosome)
o oriT: cis-acting element located at the
beginning of the transfer region and the
location where the transfer of the F
plasmid is initiated
- large transfer region with 40 genes
- Episome that harbours the F factor exists as an
independent plasmid or integrated into the
bacterial cell’s genome
- dark green regions contain genes responsible for replication and segregation of the F
episome
- light green tra regions contain genes needed for conjugative transfer
STRUCTURE OF F EPISOME
OriT (Origin of Transfer): Sequence which marks the starting point of conjugative transfer
OriV (Origin of Vegetative Replication): sequence starting with which the plasmid-DNA will be
replicated in the recipient cell
Tra region: Genes encoding the F-Pilus, DNA transfer process, mating pair formation,
synthesis of sex pilus
Pili: allow specific pairing to take place between donor and recipient cells (conjugation pi on
cell pairing by pili)
- Pilus contacts receptor on recipient cell and is
then retracted by disassembling its units
pulling the two cells together
- Donor and recipient cells remain in contact by
binding proteins located on outer membrane
- DNA is transferred from donor to recipient cell
through conjugation junction
IS (Insertion elements): composed of 1 IS2, 2 IS3 and 1 IS1000
, - Recombine with identical elements on the bacterial chromosome leading to integration
and the formation of different Hfr strains
o Hfr strains are regions in bacteria that possess the entire F episome integrated
into the bacterial genome through homologous combination
traA: Pilin, major subunit of the pilus
TRANSFER OF F EPISOME BY CONJUGATION
DNA synthesis by rolling circle replication necessary for DNA transfer by conjugation
1. DNA transfer triggered by cell-to-cell contact as pilus retracts
2. Pili involved in all conjugations between gram-negative bacteria
3. One strand of episode DNA is nicked and transferred to the recipient
4. Nicking enzyme TraI is encoded by the tra operon of the F episome
5. TraI unwinds strand of DNA to be transferred
6. DNA synthesis by rolling circle mechanism replaces the transferred strand in the donor
and relaxosome is formed
7. Complementary DNA strand is made in the recipient
8. Transfer of DNA through a channel (T4SS) initiated during rolling circle replication
9. At the end of the process, both donor and recipient possess complete episomes
10.Transferred DNA is converted into double-stranded form in the recipient bacterium
11.For transfer of F episome, if an F containing donor cell (F+) mates with a recipient
lacking the episome (F-), the result is two F+ cells
12.When the F episome is free, conjugation leaves the recipient bacterium with one copy
of the F episome
13.When the F episome is integrated, conjugation causes the transfer of bacterial
chromosome until the process is interrupted by random breakage of contact between
the cells
,Most genes in tra region are involved in DNA transfer and replication
At least 12 genes are required for modification and assembly of pilin in the sex pilus and the
stabilisation of the type 4 secretion system (T4SS) which allows transfer of DNA from one
bacterial cell to the next
tra region
contains
number of
genes and are arranged in a loci called tra and trb
traA encodes pilin
traI encodes relaxase
traD encodes coupling protein ATPase
traY encodes transcription factor – DNA binding
traM interacts with DNA and numerous proteins
ROLLING CIRCLE MECHANISM AND FORMATION OF THE RELAXOSOME
Relaxosome = relaxase (TraI) and accessory proteins (TraY, TraM, IHF)
I. Relaxosome forms at the oriT of the negatively supercoiled DNA
II. Relaxase (a dimer) nicks the oriT at a site called nic
III. Relaxase monomer forms covalent link with the 5’ end generated during
hydrolysis of the phosphodiester bond
IV. Relaxase unwinds the DNA
V. Relaxase-bound DNA recognised by the coupling protein TraD and with TraM
mediating the relaxosome-transferosome contact transported through the type 4
secretion system to a recipient cell
VI. Certain T4SS can also uptake DNA from the extracellular environment
VII. Rolling-circle replication (Polymerase III) occurs concomitant in donor and
recipient cell
HFR STRAIN
Hfr cells: cells with an F plasmid integrated into the chromosome
- High rates of genetic recombination between genes on the donor and recipient
chromosome
- Conjugation between an Hfr donor and an F- leads to transfer of genes from the host
chromosome
o Chromosome and plasmid form a single molecule of DNA
When rolling circle replication initiated by the F plasmid, replication
continues on the chromosome
Chromosome is also replicated and transferred
o Integration of a conjugative plasmid provides a mechanism
for mobilising a cell’s genome
- Integration of F plasmid into chromosome occurs at different IS sites
,- Different Hfr strains are possible
o Determines order and orientation of genes in E.coli
- Once integrated, the F episome no longer replicates independently
- Tra operon is still functional and the strain synthesises pili
- When conjugation is triggered, DNA transfer is initiated at the oriT
- Genes can be transferred because F episome is integrated in the bacterial chromosome
- Transfer of chromosomal DNA involved replication via rolling circle mechanism
- Partial transfer of chromosome results in recipient keeping F- phenotype
- Partial chromosome cannot replicate and is either lost or recombines with recipient
chromosome leading to a new phenotype
, RESISTANCE PLASMIDS
- Bacterial dysentery caused by Shigella treated with sulphanilamide since 1945
- 1952: ~80% shigella strains were sulphanilamide resistant
- 1955: one strain of Shigella was resistance to sulphanilamide, streptomycin,
chloramphenicol, and tetracycline
- Multiple-drug-resistance phenotype inherited as a single genetic package
- Resistance could be transmitted in an infectious manner
- Resistance could be transmitted to other shigella strains and other related species of
bacteria
- Vector carrying these resistances is self-replicating element similar to the F factor
- R factors are transferred rapidly on cell conjugation
Genetic map of resistance plasmid R100
- Pink circle shows size and the outer circle
show the location of antibiotic resistance
genes and other key functions:
o Mer, mercuric ion resistance
o Sul, sulphonamide resistance
o Str, streptomycin resistance
o Cat, chloramphenicol resistance
o Tet, tetracycline resistance
o OriT: origin of conjugative transfer
o Tra: transfer functions
o Insertion sequences (IS)
o Transposon (Tn10)
- Stringent plasmid: plasmid replication is
obligatory coupled to chromosome replication
o Low copy number
BACTERIOCINS
Class of compounds that bacteria use to fight off other
similar bacteria
- substances secreted by bacteria that inhibit the
growth of closely related bacterial strains
- Colicin EI is a type of bacteriocin encoded by the
ColE1 plasmid and inhibits the growth of E. coli and
Shigella
- Colicin E1 is a channel forming transmembrane
protein
o Pneumocins
o Pesticins
o Entercoccins
o Staphylococcins
- Relaxed plasmid: plasmid replication is not
obligatory coupled to chromosome replication
o High copy number
- ColE1: copy number 10-15
- ColE1 is inherently non-transferable (non-conjugative) by bacterial mating because the
genes coding for the “mating pair formation” are absent
MOLECULAR BIOLOGY FOR
NEUROSCIENTISTS
,EXTRACHROMOSOMAL REPLICATION
Origin of eukaryotes and their evolutionary relationship with Archaea is a major biological
question
- Argument about scenarios began in 1977
Two scenarios:
I. Classical view-three primary domains
a. Archaea and Eukarya have common ancestor
II. Two primary domains
a. Archaea and Bacteria are two primary domains, whereas Eukarya is a
secondary domain that arose from the merging of archaeon and a bacterium
PLASMIDS
Plasmids fall into two groups:
I. Conjugative
a. Promote sexual conjugation between bacterial cells resulting in a conjugative
plasmid spreading from one cell to all other cells in a bacterial culture
b. Conjugation and plasmid transfer controlled by tra genes
i. Present on conjugative plasmids but absence on non-conjugative types
II. Non-conjugative
a. Do not promote sexual conjugation
Plasmid Classification
I. Fertility/F plasmids
Carry only tra genes and have no characteristic beyond the ability to promote
conjugal transfer of plasmids
II. Resistance/R plasmids
Carry genes conferring on the host bacterium resistance to one or more
antibacterial agents
HORIZONTAL/LATERAL GENE TRANSFER
Vertical: transfer between parent and offspring normally generating new cells = produces
the next generation
Horizontal: prokaryotic organism directly transfers DNA without reproduction or pick up DNA
from the environment
Movement of genetic material between unicellular and/or multicellular organisms by
means other than reproduction
I. Bacterial Transformation
II. Bacterial Transduction
III. Bacterial Conjugation
HORIZONTAL GENE TRANSFER VIA TRANSFORMATION
a. Uptake of short fragments of naked DNA by transformable bacteria
b. 10 genes can be picked up
c. Long fragments are unstable because cell membrane has to open
, d. Bacterial transformation provided first support that NDA is genetic material in
classical experiemnts by Griffith and Avery
Conclusion: R-form bacterial transformed into a stable S form and the
transformation process required some factor form the heat killed S sample
Factor originally believed to be a protein because DNA was viewed
as a monotonous molecule of uniformity that would have no
biological specificity
In the 40s, it was revealed that the DNA was the demonstrating
factor
o Watson and Crick solidified the concept that DNA was the
genetic material
HORIZONTAL GENE TRANSFER VIA TRANSDUCTION
Transfer of DNA from one bacterium into another via bacteriophages
Bacteriophage: viruses that specifically infect bacteria
I. Generalised transduction: transfer of any gene from one bacterium to another at
low frequency
1. When a bacterial cell is infected with a phage, the lytic cycle may occur
2. During lytic infection, the enzymes responsible for packaging viral DNA into the
bacteriophage packages the host DNA accidently
= produces a transducing particle which cannot lead to viral infection because
they do not have viral DNA
, 3. On the lysis of a cell, transducing particles released along with normal virions
4. Lysate contains a mixture of normal virions and transducing particles
5. When lysate infect cells, small proportion receives transducing particles that inject the
DNA they packaged from a previous host bacterium
a. DNA cannot replicate but can recombine with the DNA of the new host
II. Specialised transduction: efficient, selective transfer of small region of bacterial
chromosome
HORIZONTAL GENE TRANSFER VIA CONJUGATION
Conjugation: mechanism of genetic transfer involving cell-to-cell contact
- Plasmid encoded mechanism
, - Conjugative plasmids use the mechanism to transfer copies of themselves to new host
cells
- Involves a donor cell and a recipient cell
- Genetic elements that cannot transfer themselves can become immobilised during
conjugation
Discovered by J. Lederberg and E. Tatum in 1946
Strain A: auxotroph for methionine (met) and biotin (bio) and
prototroph for (thr+, leu+, thi+)
Strain B: auxotroph for threonine (thr), leucine (leu-), thiamine
(thi-) and prototroph (met+, bio+)
Strain A washed and plated on agar plates made
with minimal medium (MM) that contains only
inorganic salts, a carbon source, and water
No colonies
Growth on the agar plate does not enable
backmutation to restore prototrophy for
methionine and biotin
Strain B was washed and plated on agar plates
made with minimal medium (MM)
No colonies
Backmutation cannot restore prototrophy
Strain A and B mixed in medium containing all
supplements before plating on agar with MM
Colonies at a frequency of 10-7
Suggests that recombination of genes taken place
between the genomes of the two strains to produce
the prototrophs or one cell leaked substances that
the other picked up (cross-feeding)
Is physical contact required to generate
prototroph?
- Suspension of bacterial strain unable to
synthesise certain nutrients is placed in one
arm of U-tube
- A strain genetically unable to synthesise
required metabolites is placed in the other arm
- Liquid may be transferred between the arms by
applying pressure or suction, but bacterial cells
cannot pass through
- After several hours of incubation, cells were
washed and plated on agar plated made with
MM
o No colonies were formed
o Requires direct contact for conjugation
and recombination
FERTILITY FACTOR (aka F
plasmid)
Allows genes to be transferred from one bacterium carrying the factor to another bacterium
lacking the factor by conjugation
, - Recognised that transfer only
occurred in one direction
o One cell act as a donor
o Lost its ability to transfer
genetic material
o Other cell acts as a recipient
- During bacterial gene transfer, the
recipient receives genetic information
from the donor and is thereby changed
- Fertility (ability to donate) of E.coli could be regained and lost
o Suggests that the donor ability itself is a herefitary state imposed by a fertility
factor (F)
- Strains that carry F can donate and are designated F+ (male)
- Strains that lack F cannot donate and are recipients are designated F- (female)
- F+ cells do not attach to other F+ cells
F EPISOME
Episomes are special form of plasmid that can also
traffic in and out of chromosomes
- ~100kb in length
- Two origins of replication:
o oriV: used when the plasmid is free in the
cell (one copy per bacterial chromosome)
o oriT: cis-acting element located at the
beginning of the transfer region and the
location where the transfer of the F
plasmid is initiated
- large transfer region with 40 genes
- Episome that harbours the F factor exists as an
independent plasmid or integrated into the
bacterial cell’s genome
- dark green regions contain genes responsible for replication and segregation of the F
episome
- light green tra regions contain genes needed for conjugative transfer
STRUCTURE OF F EPISOME
OriT (Origin of Transfer): Sequence which marks the starting point of conjugative transfer
OriV (Origin of Vegetative Replication): sequence starting with which the plasmid-DNA will be
replicated in the recipient cell
Tra region: Genes encoding the F-Pilus, DNA transfer process, mating pair formation,
synthesis of sex pilus
Pili: allow specific pairing to take place between donor and recipient cells (conjugation pi on
cell pairing by pili)
- Pilus contacts receptor on recipient cell and is
then retracted by disassembling its units
pulling the two cells together
- Donor and recipient cells remain in contact by
binding proteins located on outer membrane
- DNA is transferred from donor to recipient cell
through conjugation junction
IS (Insertion elements): composed of 1 IS2, 2 IS3 and 1 IS1000
, - Recombine with identical elements on the bacterial chromosome leading to integration
and the formation of different Hfr strains
o Hfr strains are regions in bacteria that possess the entire F episome integrated
into the bacterial genome through homologous combination
traA: Pilin, major subunit of the pilus
TRANSFER OF F EPISOME BY CONJUGATION
DNA synthesis by rolling circle replication necessary for DNA transfer by conjugation
1. DNA transfer triggered by cell-to-cell contact as pilus retracts
2. Pili involved in all conjugations between gram-negative bacteria
3. One strand of episode DNA is nicked and transferred to the recipient
4. Nicking enzyme TraI is encoded by the tra operon of the F episome
5. TraI unwinds strand of DNA to be transferred
6. DNA synthesis by rolling circle mechanism replaces the transferred strand in the donor
and relaxosome is formed
7. Complementary DNA strand is made in the recipient
8. Transfer of DNA through a channel (T4SS) initiated during rolling circle replication
9. At the end of the process, both donor and recipient possess complete episomes
10.Transferred DNA is converted into double-stranded form in the recipient bacterium
11.For transfer of F episome, if an F containing donor cell (F+) mates with a recipient
lacking the episome (F-), the result is two F+ cells
12.When the F episome is free, conjugation leaves the recipient bacterium with one copy
of the F episome
13.When the F episome is integrated, conjugation causes the transfer of bacterial
chromosome until the process is interrupted by random breakage of contact between
the cells
,Most genes in tra region are involved in DNA transfer and replication
At least 12 genes are required for modification and assembly of pilin in the sex pilus and the
stabilisation of the type 4 secretion system (T4SS) which allows transfer of DNA from one
bacterial cell to the next
tra region
contains
number of
genes and are arranged in a loci called tra and trb
traA encodes pilin
traI encodes relaxase
traD encodes coupling protein ATPase
traY encodes transcription factor – DNA binding
traM interacts with DNA and numerous proteins
ROLLING CIRCLE MECHANISM AND FORMATION OF THE RELAXOSOME
Relaxosome = relaxase (TraI) and accessory proteins (TraY, TraM, IHF)
I. Relaxosome forms at the oriT of the negatively supercoiled DNA
II. Relaxase (a dimer) nicks the oriT at a site called nic
III. Relaxase monomer forms covalent link with the 5’ end generated during
hydrolysis of the phosphodiester bond
IV. Relaxase unwinds the DNA
V. Relaxase-bound DNA recognised by the coupling protein TraD and with TraM
mediating the relaxosome-transferosome contact transported through the type 4
secretion system to a recipient cell
VI. Certain T4SS can also uptake DNA from the extracellular environment
VII. Rolling-circle replication (Polymerase III) occurs concomitant in donor and
recipient cell
HFR STRAIN
Hfr cells: cells with an F plasmid integrated into the chromosome
- High rates of genetic recombination between genes on the donor and recipient
chromosome
- Conjugation between an Hfr donor and an F- leads to transfer of genes from the host
chromosome
o Chromosome and plasmid form a single molecule of DNA
When rolling circle replication initiated by the F plasmid, replication
continues on the chromosome
Chromosome is also replicated and transferred
o Integration of a conjugative plasmid provides a mechanism
for mobilising a cell’s genome
- Integration of F plasmid into chromosome occurs at different IS sites
,- Different Hfr strains are possible
o Determines order and orientation of genes in E.coli
- Once integrated, the F episome no longer replicates independently
- Tra operon is still functional and the strain synthesises pili
- When conjugation is triggered, DNA transfer is initiated at the oriT
- Genes can be transferred because F episome is integrated in the bacterial chromosome
- Transfer of chromosomal DNA involved replication via rolling circle mechanism
- Partial transfer of chromosome results in recipient keeping F- phenotype
- Partial chromosome cannot replicate and is either lost or recombines with recipient
chromosome leading to a new phenotype
, RESISTANCE PLASMIDS
- Bacterial dysentery caused by Shigella treated with sulphanilamide since 1945
- 1952: ~80% shigella strains were sulphanilamide resistant
- 1955: one strain of Shigella was resistance to sulphanilamide, streptomycin,
chloramphenicol, and tetracycline
- Multiple-drug-resistance phenotype inherited as a single genetic package
- Resistance could be transmitted in an infectious manner
- Resistance could be transmitted to other shigella strains and other related species of
bacteria
- Vector carrying these resistances is self-replicating element similar to the F factor
- R factors are transferred rapidly on cell conjugation
Genetic map of resistance plasmid R100
- Pink circle shows size and the outer circle
show the location of antibiotic resistance
genes and other key functions:
o Mer, mercuric ion resistance
o Sul, sulphonamide resistance
o Str, streptomycin resistance
o Cat, chloramphenicol resistance
o Tet, tetracycline resistance
o OriT: origin of conjugative transfer
o Tra: transfer functions
o Insertion sequences (IS)
o Transposon (Tn10)
- Stringent plasmid: plasmid replication is
obligatory coupled to chromosome replication
o Low copy number
BACTERIOCINS
Class of compounds that bacteria use to fight off other
similar bacteria
- substances secreted by bacteria that inhibit the
growth of closely related bacterial strains
- Colicin EI is a type of bacteriocin encoded by the
ColE1 plasmid and inhibits the growth of E. coli and
Shigella
- Colicin E1 is a channel forming transmembrane
protein
o Pneumocins
o Pesticins
o Entercoccins
o Staphylococcins
- Relaxed plasmid: plasmid replication is not
obligatory coupled to chromosome replication
o High copy number
- ColE1: copy number 10-15
- ColE1 is inherently non-transferable (non-conjugative) by bacterial mating because the
genes coding for the “mating pair formation” are absent
