Transcript Inquiry into Life Twelfth Edition

Lecture PowerPoint to accompany
Molecular Biology
Fourth Edition
Robert F. Weaver
Chapter 23
Transposition
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
23.1 Bacterial Transposons
• A transposable element moves from one
DNA address to another
• Originally discovered in maize,
transposons have been found in all kinds
of organisms
– Bacteria
– Plants
– Humans
23-2
Discovery of Bacterial
Transposons
•
•
•
•
•
Phage coat is made of protein
Always has the same volume
DNA is much denser than protein
More DNA in phage, denser phage
Extra DNAs that can inactivate a gene by
inserting into it were the first transposons
discovered in bacteria
• These transposons are called insertion
sequences (ISs)
23-3
Insertion Sequences
• Insertion sequences are the simplest type of
bacterial transposon
• They contain only the elements necessary for
their own transposition
– Short inverted repeats at their ends
– At least 2 genes coding for an enzyme, transposase
that carries out transposition
• Transposition involves:
– Duplication of a short sequence in the target DNA
– One copy of this sequence flanks the insertion
sequence on each side after transposition
23-4
Generating Host DNA Direct
Repeats
23-5
Complex Transposons
• The term “selfish DNA” implies that
insertion sequences and other
transposons replicate at the expense of
their hosts, providing no value in return
• Some transposons do carry genes that are
valuable to their hosts, antibiotic
resistance is among most familiar
23-6
Antibiotic Resistance and
Transposons
• Donor plasmid has
Kanr, harboring
transposon Tn3 with
Ampr
• Target plasmid has Tetr
• After transposition, Tn3
has replicated and
there is a copy in target
plasmid
• Target plasmid now
confers both Ampr, Tetr
23-7
Transposition Mechanisms
• Transposons are sometimes called “jumping
genes”, DNA doesn’t always leave one place for
another
• When it does, nonreplicative transposition
– “Cut and paste”
– Both strands of original DNA move together from 1
place to another without replicating
• Transposition frequently involves DNA
replication
–
–
–
–
1 copy remains at original site
New copy inserts at the new site
Replicative transposition
“Copy and paste”
23-8
Replicative Transposition of Tn3
• In first step, 2 plasmids fuse, phage replication, forms a
cointegrate – coupled through pair of Tn3 copies
• Next is resolution of cointegrate, breaks down into 2
independent plasmids, catalyzed by resolvase gene
product
23-9
Detailed Tn3 Transposition
23-10
Nonreplicative Transposition
• Starts with same 2 first
steps as in replicative
transposition
• New nicks occur at arrow
marks
• Nicks liberate donor
plasmid minus the
transposon
• Filling gaps and sealing
nicks completes target
plasmid and its new
transposon
23-11
23.2 Eukaryotic Transposons
• Transposons have powerful selective
forces on their side
• Transposons carry genes that are an
advantage to their hosts
– Their host can multiply at the expense of
completing organisms
– Can multiply the transposons along with rest
of their DNA
• If transposons do not have host
advantage, can replicate themselves
within their hosts
23-12
Examples of Transposable
Elements
• Variegation in the color of maize kernels is
caused by multiple reversions of an
unstable mutation in the C locus,
responsible for kernel color
• Mutation and its reversion result from Ds
(dissociation) element
– Transposes into the C gene
– Mutates it
– Transposes out again, revert to wild type
23-13
Ds and Ac of Maize
• Ds cannot transpose on its own
• Must have help from an autonomous
transposon, Ac (for activator)
– Ac supplies transposase
– Ds is an Ac element with most of its middle
removed
– Ds needs
• A pair of inverted terminal repeats
• Adjacent short sequences that Ac transposase can
recognize
23-14
Transposable Elements in Maize
23-15
Structures of Ac and Ds
23-16
P Elements
• The P-M system of hybrid dysgenesis in
Drosophila is caused by conjunction of 2 factors:
– Transposable element (P) contributed by the male
– M cytoplasm contributed by the female allows
transposition of the P element
• Hybrid offspring of P males and M females suffer
multiple transpositions of P element
• Damaging chromosomal mutations are caused
that render the hybrids sterile
• P elements have practical value as mutagenic
and transforming agents in genetic experiments
with Drosophila
23-17
23.3 Rearrangement of
Immunoglobulin Genes
• Mammalian genes use a process that
closely resembles transposition for:
– B cell antibodies
– T cell receptors
• Recombinases involved in these
processes have similar structures
23-18
Antibody Structure
• Antibody is composed
of 4 polypeptides
– 2 heavy chains
– 2 light chains
• Sites called variable
regions
– Vary from 1 antibody
to another
– Gives proteins their
specificity
• Rest of protein is
constant region
23-19
Immune System Diversity
• Enormous diversity of immune system is
generated by 3 basic mechanisms:
– Assembling genes for antibody light chains
and heavy chains from 2 or 3 component
parts
– Joining the gene parts by an imprecise
mechanism that can delete bases or add
extra bases
– Causing a high rate of somatic mutations,
probably during proliferation of a clone if
immune cells
23-20
Rearrangement of Antibody
Light Chain Gene
23-21
Antibody Heavy Chain Coding
Regions
Human heavy chain is encoded in
–
–
–
–
48 variable segments
23 diversity segments
6 joining segments
1 constant segment
23-22
Recombination Signals
• The recombination signal sequences
(RSSs) in V(D)J recombination consist of:
– Heptamer
– Nonamer
– Separated by 12-bp or 23-bp spacers
• Recombination occurs only between a 12
signal and a 23 signal
• Guarantees that only 1 of each coding
region is incorporated into the rearranged
gene
23-23
The Recombinase
• Recombination-activating gene (RAG-1)
stimulated V(D)J joining activity in vivo
• Another gene tightly liked to RAG-1 also
works in V(D)J joining, RAG-2
• These genes, RAG-1 and RAG-2, are
expressed only in pre-B and pre-T cells
23-24
Mechanism of V(D)J
Recombination
• RAG1 and RAG2 introduce single-strand nicks
into DNA adjacent to either a 12 signal or 23
signal
• Results in transesterification where newly
created 3’-OH group:
– Attacks the opposite strand
– Breaks it
– Forms hairpin at the end of the coding segment
• Hairpins then break in an imprecise way that
allows joining of coding regions with loss of
bases or gain of extra bases
23-25
23.4 Retrotransposons
• Retrotransposons replicate through an
RNA intermediate
• Retrotransposons resemble retroviruses
– Retroviruses can cause tumors in vertebrates
– Some retroviruses cause diseases such as
AIDS
• Before studying retrotransposons, look at
replication of the retroviruses
23-26
Retroviruses
• Class of virus is named for its ability to
make a DNA copy of its RNA genome
• This reaction is the reverse of the
transcription reaction – reverse
transcription
• Virus particles contain an enzyme that
catalyzes reverse transcription reaction
23-27
Retrovirus Replication
• Viral genome is RNA,
with long terminal repeats
at each end
• Reverse transcriptase
makes linear, ds-DNA
copy of RNA
• ds-DNA copy integrates
back into host DNA =
provirus
• Host RNA polymerase II
transcribes the provirus to
genomic RNA
• Viral RNA packaged into
a virus particle
23-28
Model for Synthesis of Provirus
DNA
• RNase H degrades the
RNA parts of RNA-DNA
hybrids created during
the replication process
• Host tRNA serves as
primer for reverse
transcriptase
• Finished ds-DNA copy of
viral RNA is then inserted
into the host genome
• It can be transcribed by
host polymerase II
23-29
Retrotransposons
• Several eukaryotic transposons transpose in a
way similar to retroviruses
– Ty of yeast
– copia of Drosophila
• Start with DNA in the host genome
– Make an RNA copy
– Reverse transcribe it within a virus-like particle into
DNA that can insert into new location
• HERVs likely transposed in the same way until
ability to transpose lost
– HERV = human endogenous retroviruses
23-30
Ty Transcription
23-31
Non-LTR Retrotransposons
• LTR are lacking in most retrotransposons
• Most abundant type lacking LTR are LINEs and
LINE-like elements
– Long interspersed elements
– Encode an endonuclease that nicks target DNA
– Takes advantage of new DNA 3’-end to prime reverse
transcriptase of element RNA
– After 2nd strand synthesis, element has been
replicated at target site
• New round of transposition begins when the
LINE is transcribed
• LINE polyadenylation signal is weak, so
transcription of a LINE often includes exons of
downstream host DNA
23-32
Nonautonomous
Retrotransposons
• Nonautonomous retrotransposons include
very abundant human Alu elements and
similar elements in other vertebrates
• Cannot transpose by themselves as they
do not encode any proteins
• Take advantage of retrotransposition
machinery of other elements such as LINE
• Processed pseudogenes likely arose in
same manner
23-33
Group II Introns
• Group II introns
– Retrohome to intronless copies same gene by:
• Insertion of an RNA intron into the gene
• Followed by reverse transcription
• Then second-strand synthesis
– Retrotranspose by:
• Insertion of an RNA intron into an unrelated gene
• Target-primed reverse transcription
• Lagging-strand DNA fragments as primers
• Group II retrotransposition:
– Forerunner of eukaryotic spliceosomal introns
– Accounted for appearance in higher eukaryotes
23-34