5BBG0204 Human and Molecular Genetics B (5BBG0204)
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In-depth notes on 5BBG0204 Human and Molecular Genetics B containing all detail mention in class and extra
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5BBG0204 Human and Molecular Genetics B (5BBG0204)
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Kings College London (KCL)
In-depth notes on 5BBG0203 Human and Molecular Genetics A containing all detail mention in class and extra. Notes written after rewatching lecture and details were added when content wasn't clear. FAQ were asked when not sure and responses were added on to the lecture notes
5BBG0204 Human and Molecular Genetics B (5BBG0204)
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L01/L02: transposons and retroelements
Transposons are discrete DNA sequences in the genome that can be mobile. They
are found in both prokaryotes and eukaryotes
• There are two main classes of transposons: Class I transposons and Class II
transposons
• Class I transposons are also called retrotransposons. They move via reserve
transcription and RNA intermediates. Often called ‘copy and paste’
transposable elements as they leave original copy in the original sequence
• Class II transposons are often called DNA transposons. They move via a
protein called transposase. Often called ‘cut and paste’ transposable
elements as sequence are cut of the original sequence
In prokaryotes only DNA transposons (Class II) carry additional genes that are not in
volved in the movement
Discovery of Class II transposons (DNA transposons)
o Barbara McClintock- in maize by looking at the purple pigment in maize
o Bacterial geneticist - found an usual type of polar mutation in bacterial
operons which appeared to revert to wild type spontaneous
▪ Polar mutation – mutation in one gene that affects expression of
other downstream genes in the same operon usually by
producing premature termination of transcription/translation
resulting in truncated mRNA
▪ On type of polar mutation is caused by insertion of considerable
length of DNA
▪ The scientists found insertion elements (IS) which caused polar
mutations
• Insertion elements are DNA transposons found in E.coli that were first
detected by Saedler, Starling and Shapiro. First IS element found was the gal
operon in E.coli which are three genes involved in the metabolism of the sugal
galactose
• When there an IS in the middle it results truncated mRNA and massive
frameshifts, hence. Only one enzyme is produced
• Physical demonstration of insertion sequences
o Caesium chloride gradient centrifugation was used to compare DNA
from phage lambda particles which carry the E. coli gene for
galactosidase gal+ and gal-. The λgal- has a heavier density due to
the presence of an insertion sequence.
▪ Pieces of DNA that are different sizes
▪ Place them in a tube and centrifuge them forming a gradient
▪ DNA will bind on the gradient depending on size
▪ Phage lambda integrates into the genome between the gal and
bio operons of the E. coli genome. Sometimes when it excises
out of the of the E. coli genome it takes either some of bio or gal
operon with it. Called specialised transduction.
, • bacteriophage lambda inserts next to the gal operon in E. coli. Researchers
took lambda carrying the wild type gal operon (λgal+) and lambda containing
a mutant form of gal (λgal-) produced by an insertion sequence. They
denatured the two different lambda DNA by boiling at 100°C and then let the
two forms renature to form a heteroduplex. These experiments showed a
single stranded loop of λgal- showing the position of the IS element (arrow).
•
• IS are typically 800 and 2000 bp
• IS have inverted DNA repeats at their ends
• The central part consists of an open reading frame which codes transposase
responsible for transcription
• IS1 first identified in E.coli’s galactose operon is present in 5-8 copies in the
E.coli chromosome
o IS1 is 768 bp long, 23 bp long inverted terminal repeats
o IS1 carries a single gene coding for a transposase
o Produces a 9bp duplication of the insertion site
• Since there are IS in the F plasmid and IS sequence on E.coli genome, they
have regions of homology and therefore there’s homologous recombination
allowing the F plasmid to integrate to the E.coli genome and create a Hfr
strain
• There are 3 types of DNA transposons classified by structure in prokaryotes
o IS elements- have inverted repeats at ends and only carry transposase
gene
o Composite DNA transposons- carry additional genes but originally
derived from two IS elements. The additional genes are usually genes
for antibiotic resistance
o Nanocomposite DNA transposons- carry additional genes as weel as
transposase genes. The additional genes are usually genes for
antibiotic resistance
• Unlike prokaryotic transposons, eukaryotic DNA transposons do not carry
additional gene they only have the transposase gene
• Believed that there was an evolutionary pressure for transposons to acquire
antibiotic resistance markers and to move around and therefore share the
antibiotic resistance around- reason why additional genes are not found in
eukaryotes
• Composite DNA transposons carry genes flanked on both sides of IS
elements. An example is Tn10
• Tn10 is a 9.3kb and includes 6.5 kb of central DNA (includes a gene for
tetracycline resistance) and 1.4kb inverted IS elements. IS elements supply
transposase and ITR recognition signals
• Tn10 has two IS10 elements. These IS10 elements can’t live independently,
they are locked in with the tetracycline resistance genes. This is because of
the mutations they have accumulated
• Due to the mutations on the two IS10 elements, they aren’t recognised by
transposase, therefore they don’t move around. Therefore transposase can
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, only use the IS located at the ends of the sequences, to move the transposon
from one position to another
• Non composite transposons carry genes but do not terminate with IS
elements only have inverted repeated sequences at the end. An example is
Tn3
• Tn3 is 5kb in length with 38bp ITR and includes 3 genes: AM R (ampicillin
resistance gene), tnpA (transposase) and tnpR (resolvase, which functions in
recombination)
• Methods of transposition: non replicative transposition and replicative
transposition.
• Non replicative transposition is how the majority of DNA transposons move
with exception being Tn3. Eukaryotic DNA transposons also uses non
replicative transposition
• Non replicative transposition requires transposase which all transposons carry
the gene for. T
• Non replicative transposition initiates when transposase recognises the
inverted terminal repat at the ends of the IS.
• Transposase will cut the transposon out of the original site and move it to
another site called the target site. (DNA transposons are class II transposons
therefore they are cut and paste
• Non replicative transposition mechanism:
o 1. transposase makes blunt end cut in the donor DNA and staggered
cuts in the target DNA
o 2. transposase ligates the transposon to 5’ single stranded ends of
target DNA
o 3. cellular DNA polymerase extends the 3’ cut ends and DNA ligase
joins the ends. Due to the staggered cut in the target site, there’s direct
repeats on the genome
• The non-replicative transposition mechanism is mediated by two molecules of
transposase acting together. As each transposase will bind to each inverted
repeat
o Each transposase binds to the inverted repeats at the end of the
transposon
o The two transposases' molecules form a dimmer, and cut the
transposons form the DNA donor site
o The transposase molecules are involved in staggered cutting and
inserting the transposon in the DNA target site
• Transposase doesn’t recognise a particular sequence, it does it relatively
randomly in the genome
• Replicative transposition is used by Tn3. This is unusual because it creates
another copy of the transposon at a different location in the genome.
• Replicative transposition mechanism:
o 1. two closed circular DNAs (donor with Tn3 and target).
Recombination between the two DNAs (catalysed by transposase, Tn3
gene tnpA) to form cointegrate. This is an example of illegitimate
recombination because requires little if any sequence similarity
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, o 2. Resolution of cointegrate in which cointegrate breaks down into two
independent DNAs each bearing one copy of Tn3
▪ Catalysed by resolvase (coded for by tnpR gene)
▪ Recombination is between two homologous sites on Tn3 called
res sites
▪ TnpR protein (resolvase) also functions as repressor of tnpA
and tnpR which limits the amount of transposase available and
therefore prevents the transposon moving to frequently
• It's not in transposon best interest to move, as it can land in a gene that is
essential for cell’s survival. Therefore transposition in controlled by reducing
the levels of transposase
• There are multiple mechanisms to reduce transposition:
o Inefficient/weak promoter- limits transcription which low/undetectable
amounts of transposase mRNA and therefore means there are low
levels of transposase. Example: IS21 has no detectable transposase
mRNA
o Promoters are located in the terminal inverted repeats so binding of the
transposase or truncated derivatives can block transcription. Example:
IS1 regulator protein InsA in the terminal transposase binding domain
o Some transposons have alternative promoters which produce a
truncated version of transposase which inhibits transposase activity.
Example: IS50
o Some transposons produce unstable mRNA which is degraded quickly.
Example: IS5
o Some transposons produce a small antisense RNA which binds to the
normal transposase mRNA. Example: IS10 and Tn10
o Some transposons have been shown to affected by methylation.
▪ Promoter methylation turns off transcription. Example: Tn10-
dam methylation of Pin promoter decreases RNA polymerase
binding
▪ Methylation of the inverted repeats makes them less able to bind
to transposase. Example: Tn10
• Tn10 knocks down the transpose gene by producing a short antisense RNA.
The normal transposase mRNA is produced from the Pin promoter and the
short antisense RNA is produced from Pout promoter
• TN10- The antisense RNA binds to the sense mRNA forming a double
stranded RNA molecule and prevents translation of the mRNA by the
ribosome into transposase protein
• No transpose= no jumping
• The frequency of transposition is 1 x 10^-3 to 1 x 10 ^-4 per element per
generation – very low
• The chance an IS element will insert into one gene in E.coli genome is 1 x
10^-5 to 1 x 10^-7 per generation
• The chance of reversion (change of an IS element leaving gene) is 1 x 10^-6
to 1 x 10^-10
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