1. Molecular Biology Fourth 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.

2. 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

3. 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)

4. 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

5. Generating Host DNA Direct Repeats

6. 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

7. 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

8. 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”

9. 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

10. Detailed Tn3 Transposition

11. 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

12. 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

13. 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

14. 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

15. Transposable Elements in Maize

16. Structures of Ac and Ds

17. 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

18. 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

19. 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

20. 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

21. Rearrangement of Antibody Light Chain Gene

22. Antibody Heavy Chain Coding Regions
Human heavy chain is encoded in
48 variable segments
23 diversity segments
6 joining segments
1 constant segment

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. Ty Transcription

32. 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

33. 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

34. Group II Introns Group II introns Group II retrotransposition:
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