2. Transposable elements
• “mobile genetic elements”
• comprise 45% of human chromosomal DNA
“middle repetitive DNA”
• contribute to spontaneous mutation, genetic
rearrangements, horizontal transfer of genetic
material
• aid speciation and genomic change (in bacteria
transposons are often associated with antibiotic
resistance genes)
• cells must depress transposition to insure genetic
stability
3. Types of transposable elements
• DNA vs. RNA
• viral vs. nonviral
• replicative mechanism vs. excision
mechanism
6. Discovery of transposons
• Barbara McClintock 1950’s Ac Ds system in
maize influencing kernel color
unstable elements
changing map position
promote chromosomal breaks
• Rediscovery of bacterial insertion sequences
source of polar mutations
discrete change in physical length of DNA
inverted repeat ends: form “lollipops” in EM after
denaturation/reannealing
7. Composite bacterial transposons
• repeated ends, usually inverted, sometimes
direct
• repeated ends themselves are IS elements
and can independently transpose
• ends mobilize all intervening DNA
• often antibiotic resistance genes (examples
Tn3 (ampicillin), Tn5 (kanamycin), Tn10
(tetracycline)
• often reside on plasmids
8. Basic minimal insertion sequence structure
ends: genetically required, in cis
tnp (transposase): genetically required, trans-acting
tnp ORF
Element 1 Element 2 Transposition?
wt wt 1 and 2
ends- ends- neither
trp- tnp- neither
ends- wt only 2
tnp- wt 1 and 2
tnp- ends- only 1
10. tnpR and res mutations cause
accumulation of “co-integrate structure”
direct repeat of Tn
cointegrate
11. 2 types of DNA tranposons
• excisive mechanism
examples: Tn5, Tn10, P elements
• replicative mechanism
examples: Tn3, bacteriophage Mu
12. Replicative transposons
• orignal cut of transposon is only nick and
only one strand is initially ligated
• element replicates through itself
• produces as intermediate a “co-integrate”
structure
• co-integrate is resolved by resolvase (as
TnpR of Tn3) and at specific site (as res of
Tn3)
13. Excisive transposons
• cut-and-paste mechanism
• cut themselves out of original site, producing
double strand break
• cut target site and ligate to element ends, thereby
inserting at new site
• original site break repaired
usually with sister chromosome, restoring
transposon at original site
sometimes end healed without transposon, can
also be associated with deletion at excision site
14. Source of target site duplication
“TSD”
GAC
CTG
CTG
GAC
Staggered cleavage of target
CTG
GAC
Ligation of transposon DNA
GAC
CTG
Repair replication generates short direct repeats
15. “degenerate” transposons
• many naturally occurring transposable elements
have suffered mutation and are no longer active
• some of these may have cis-acting end mutations
and cannot be mobilized
• others may have intact ends but no transposase:
these can be mobilized by a element that is tnp+
(“autonomous” element)
• Ac Ds system is an example of latter: Ac
(activator) can mobilize Ds (dissociator)
• MITEs (minature inverted repeat transposable
elements) are nonautonomous DNA elements
• SINEs are retrotransposon version (LINEs)
16. Comparison of transposition reactions
Direct transesterification reactions DDE motif transposase (integrase)
20. Classification of retroelements
• Have obligate RNA intermediate, use
reverse transcriptase (RT, RNA-dependent
DNA polymerase)
• LTR-retroelements: long terminal repeats
Ty1/copia, Ty3/gypsy, retroviruses
• Non-LTR-retroelements “retroposons”
LINES
21.
22. Characteristics of LTR
retroelements
• Long terminal repeats: required for replication
cycle
• Genes: gag, pol, (viruses also have env)
• Pol is polyprotein which gives rise to RT (reverse
transcriptase), IN (integrase) RH (RNase H), PR
(protease)
• Forms VLPs virus-like particles
• Integrase is functionally and structurally similar to
transposase of DNA transposons, DDE motif
• Integration gives characteristic TSD
29. Human L1 (LINE-1)
retroelement
• 15% of human DNA
• 520,000 copies, only 3-5,000 are full-length
• Associated with human disease loci
• Transpose specifically in germ line
