Bacterial insertion sequences
Bacterial insertion sequences Hybridization experiments for gal- insertion mutants found that same sequence over & over again In various gal- alleles In other parts of the bacterial chromosome
Found other repeated Insertion Sequences through similar hybridization experiments
T Western Biol 202
1
Bacterial insertion sequences Structure: Encodes transposase protein (performs transposition) Terminal inverted repeats (required for transposition) Target site duplication (created during insertion)
EG 21.1
T Western Biol 202
2
Bacterial transposons Composite Bacterial genes flanked by 2 Insertion Sequences Depending on transposon, both IS’s could move on own… OR one IS may be disabled & transposition depends on the intact IS See also Fig 14-6a
EG 21-2,3
T Western Biol 202
3
Bacterial transposons Simple No insertion sequences Flanked by inverted repeats Encodes transposase
Tn3
Fig 14-6b T Western Biol 202
4
Mechanisms of transposition Conservative – “cut & paste” into new location e.g. Tn10
Replicative – adds new copy e.g. Tn3
Fig 14-9 T Western Biol 202
5
Conservative transposition Transposasemediated Cuts at target site Cuts out from old site Inserts in new site Host repairs cut sites to generate target site duplication e.g. Tn10
Fig 14-8 T Western Biol 202
6
Replicative transposition
Fig 14-10 T Western Biol 202
7
Medical consequences of bacterial transposons
Multiresistance plasmids
Fig 14-7 T Western Biol 202
8
Medical consequences of bacterial transposons Multiresistance plasmids
Form through: transposition into plasmid Recombination between IS’s T Western Biol 202
9
Medical consequences of bacterial transposons Multiresistance plasmids Can easy pass between bacteria by conjugation Not limited by species
Fig 14-10
Fig 5-8 T Western Biol 202
10
Eukaryotic transposable elements 2 types:
Class I - Retrotransposons LTR retrotransposons LTR
GAG
POL
LTR
Non-LTR retrotransposons LINEs -polyA SINEs 5’UTR
ORF1
ORF2
3’UTR
-polyA
Class II ‒ DNA transposons TIR
transposase
(ORF2)
TIR
N. Juretic T Western Biol 202
11
Retroviruses & retrotransposons Retroviruses RNA genome
DNA copy with reverse transcriptase Insert into host genome for multiplication Fig 14-11 T Western Biol 202
12
Retroviruses & retrotransposons Features of a retrovirus
Long terminal [direct] repeats (LTR) gag – structural protein of capsid pol – reverse transcriptase env – protein of virus envelope
Fig 14-12a T Western Biol 202
13
Retroviruses & retrotransposons Retrotransposons resemble a retrovirus without the env gene
Fig 14-12b,c
So, is a retrovtransposon a failed virus OR Is a virus an escaped retrotransposon? T Western Biol 202
14
Transposition of retrotransposons
Much like retrovirus with DNA copy returned to nucleus & inserted back into the genome Rather than making many RNA copies that are packaged T Western Biol 202
Fig 14-13
15
Transposition of retrotransposons Proof of RNA intermediate Ty1 element of yeast Engineered Ty1 on plasmid inducible by galactose Engineered Ty1 contains intron When induce transcription got increased transposition New inserts of Ty1 lacked intron! Fig 14-14
T Western Biol 202
16
Variations on retrotransposons Drosophila copia elements – “standard” gypsy elements still have env!
Fig 13-15c
Humans
Long Interspersed Elements (LINEs) & SINEs Atypical – not organized like standard retrotransposon Still has pol LINEs are autonomous & SINEs are nonautonomous
T Western Biol 202
Fig 14-20
17
Return to nucleus as RNA with 2 encoded proteins
Transposition of LINEs (L1)
ORF2 = endonuclease to cut DNA + reverse transcriptase to copy RNA into DNA
Cytoplasm
Nucleus T Western Biol 202
18
Eukaryotic transposable elements 2 types:
Class I - Retrotransposons LTR retrotransposons LTR
GAG
POL
LTR
Non-LTR retrotransposons LINEs -polyA SINEs 5’UTR
ORF1
ORF2
3’UTR
-polyA
Class II ‒ DNA transposons TIR
transposase
(ORF2)
TIR
N. Juretic T Western Biol 202
19
Barbara McClintock’s “controlling elements” are DNA transposons
Ds is nonautonomous Requires Ac to move
Ac is autonomous
T Western Biol 202
Fig 14-4
20
Barbara McClintock’s “controlling elements” are DNA transposons Ac/Ds of maize Ds elements are Ac elements with internal deletions that lead to loss of transposase function
Other families of autonomous/ nonautonomous transposable elements in maize E.g. En/Spm
T Western Biol 202
21
Barbara McClintock’s “controlling elements” are DNA transposons Transposition mechanism of Ac All done by transposase
Transposons can leave “footprints” if recombination is imprecise Will become stable mutation rather than return to wildtype condition T Western Biol 202
Fig 14-18
22
Hybrid dysgenesis is the result of DNA transposons (P elements) Crosses of wild isolate males (P) x lab strain females (M) Female (wild)
F1 progeny sterile – chromosome breakage, unstable mutations in germ line Unstable mutations of white from dysgenic cross reminscent of what seen before…
Fig 14-15
Male (lab stock)
T Western Biol 202
23
Hybrid dysgenesis is the result of DNA transposons (P elements) P elements of Drosophila Are also nonautonomous versions with internal deletions
T Western Biol 202
Fig 14-16
24
Hybrid dysgenesis is the result of DNA transposons (P elements) Hybrid dysgenesis explained: P cytotype (wild) strains have a repressor of P element transposition in germline cells M cytotype (lab) strains do not so P can transpose Since mother contributes cytoplasm to zygote, only a problem with M females
T Western Biol 202
Fig 14-17
25
Hybrid dysgenesis is the result of DNA transposons (P elements) P-repressor & Ptransposase encoded by alternative splicing: Somatic cells – intron 3 unspliced repressor Germ cells – all introns spliced transposase
EG 21-24
T Western Biol 202
26
Hybrid dysgenesis is the result of DNA transposons (P elements)
In P cytotype females, Prepressor deposited into oocytes, so “immune”
EG 21-25
T Western Biol 202
27
Eukaryotic transposable elements 2 types:
Class I - Retrotransposons LTR retrotransposons LTR
GAG
POL
LTR
Non-LTR retrotransposons LINEs -polyA SINEs 5’UTR
ORF1
ORF2
3’UTR
-polyA
Class II ‒ DNA transposons TIR
transposase
(ORF2)
TIR
N. Juretic T Western Biol 202
28
Harnessing of eukaryotic transposable elements P elements as a molecular biology tool Insertional mutagenesis Let transposons “jump” & isolate rare stable mutants Mutants created by insertional mutagenesis have a “tag” of known DNA sequence so easy to clone affected genes (often called “transposon tagging”) T Western Biol 202
Fig 14-17
29
Harnessing of eukaryotic transposable elements
P elements for Drosophila transformation
Fig 14-19
Use to “transform” flies with particular genes P element used as carrier to insert a gene into the chromosomes of germ cells of the dysgenic flies Progeny will reflect the expression of that gene T Western Biol 202
30
Transposable elements & host genomes DNA sequences that can move and multiply within the genome selfish DNA
self-replication: necessary and sufficient explanation
Explanation for the C-value Paradox
N. Juretic T Western Biol 202
31
Transposons & genome size C value paradox Genome size doesn’t correlate with complexity of organism or with gene number Organism
Genome size (Mbp)
Number predicted genes
Yeast
12
6,144
Arabidopsis
125
25,706
Rice
400
37,500
Maize
2500
50,000
C. elegans
103
20,598
Drosophila
170
13,525
Pufferfish
329
22,089
Humans
3223
~24,000 T Western Biol 202
32
Transposons & genome size Larger genome, more transposons… Organism
Genome size (Mbp)
Number predicted genes
Percent genome is transposons
Yeast
12
6,144
Arabidopsis
125
25,706
10.5%
Rice
400
37,500
35%
Maize
2500
50,000
80%
C. elegans
103
20,598
6.5%
Drosophila
170
13,525
3.1%
Pufferfish
329
22,089
2.7%
Humans
3223
~24,000
45% T Western Biol 202
33
Transposons & genome size Larger genome, more transposons…
Fig 14-22 T Western Biol 202
34
Transposons & the human genome ~45% of genome is transposable elements Figs 14-20
Fig 14-21
T Western Biol 202
35
Transposable elements & host genomes DNA sequences that can move and multiply within the genome selfish DNA
self-replication: necessary and sufficient explanation
How do these selfish elements avoid killing their hosts? If host dies or goes extinct, no transposable elements
N. Juretic T Western Biol 202
36
How can so many transposons be maintained? Fig 14-15
Transposon graveyard
Female (wild)
Male (labstock)
most are “dead” Lost transposase or mutations to flanking repeats
Inactive transposons Kept off by transposon repressors Tend to activate under stress conditions T Western Biol 202
37
How can so many transposons be maintained? Inconspicuous Found in between genes & in introns
Fig 14-21
T Western Biol 202
38
How can so many transposons be maintained? Inconspicuous Found in between genes & in introns Insert into each other (looks like just one to genome…)
Nearly all RTE insertions are into even older RTEs. A portion of a maize chromosome is illustrated. (Walbot et al 2001 PNAS) T Western Biol 202
39
How can so many transposons be maintained? Inconspicuous Found in between genes & in introns Insert into each other (looks like just one to genome…) Safe havens
T Western Biol 202
40 Fig 14-23b
How can so many transposons be maintained? Host is able to inactivate Often found in heterochromatin – kept off by methylation, etc Other host mechanisms related to those used to suppress virus replication
Fig 13-29 T Western Biol 202
41
Transposable elements & host genome evolution DNA sequences that can move and multiply within the genome selfish DNA
self-replication: necessary and sufficient explanation
Transposable elements can be harnessed by their hosts for genomic elements & genome evolution
N. Juretic T Western Biol 202
42
Transposable elements & genome structure Useful Structural role around centromeres? Telomeres of Drosophila - composed of retrotransposons – HeT-A & TART
T Western Biol 202
43
Transposable elements & genome structure Mediate genome evolution? New combinations of genes or gene elements through recombination between homologous transposable elements
T Western Biol 202
44
Found other repeated Insertion Sequences through similar hybridization experiments
T Western Biol 202
1
Bacterial insertion sequences Structure: Encodes transposase protein (performs transposition) Terminal inverted repeats (required for transposition) Target site duplication (created during insertion)
EG 21.1
T Western Biol 202
2
Bacterial transposons Composite Bacterial genes flanked by 2 Insertion Sequences Depending on transposon, both IS’s could move on own… OR one IS may be disabled & transposition depends on the intact IS See also Fig 14-6a
EG 21-2,3
T Western Biol 202
3
Bacterial transposons Simple No insertion sequences Flanked by inverted repeats Encodes transposase
Tn3
Fig 14-6b T Western Biol 202
4
Mechanisms of transposition Conservative – “cut & paste” into new location e.g. Tn10
Replicative – adds new copy e.g. Tn3
Fig 14-9 T Western Biol 202
5
Conservative transposition Transposasemediated Cuts at target site Cuts out from old site Inserts in new site Host repairs cut sites to generate target site duplication e.g. Tn10
Fig 14-8 T Western Biol 202
6
Replicative transposition
Fig 14-10 T Western Biol 202
7
Medical consequences of bacterial transposons
Multiresistance plasmids
Fig 14-7 T Western Biol 202
8
Medical consequences of bacterial transposons Multiresistance plasmids
Form through: transposition into plasmid Recombination between IS’s T Western Biol 202
9
Medical consequences of bacterial transposons Multiresistance plasmids Can easy pass between bacteria by conjugation Not limited by species
Fig 14-10
Fig 5-8 T Western Biol 202
10
Eukaryotic transposable elements 2 types:
Class I - Retrotransposons LTR retrotransposons LTR
GAG
POL
LTR
Non-LTR retrotransposons LINEs -polyA SINEs 5’UTR
ORF1
ORF2
3’UTR
-polyA
Class II ‒ DNA transposons TIR
transposase
(ORF2)
TIR
N. Juretic T Western Biol 202
11
Retroviruses & retrotransposons Retroviruses RNA genome
DNA copy with reverse transcriptase Insert into host genome for multiplication Fig 14-11 T Western Biol 202
12
Retroviruses & retrotransposons Features of a retrovirus
Long terminal [direct] repeats (LTR) gag – structural protein of capsid pol – reverse transcriptase env – protein of virus envelope
Fig 14-12a T Western Biol 202
13
Retroviruses & retrotransposons Retrotransposons resemble a retrovirus without the env gene
Fig 14-12b,c
So, is a retrovtransposon a failed virus OR Is a virus an escaped retrotransposon? T Western Biol 202
14
Transposition of retrotransposons
Much like retrovirus with DNA copy returned to nucleus & inserted back into the genome Rather than making many RNA copies that are packaged T Western Biol 202
Fig 14-13
15
Transposition of retrotransposons Proof of RNA intermediate Ty1 element of yeast Engineered Ty1 on plasmid inducible by galactose Engineered Ty1 contains intron When induce transcription got increased transposition New inserts of Ty1 lacked intron! Fig 14-14
T Western Biol 202
16
Variations on retrotransposons Drosophila copia elements – “standard” gypsy elements still have env!
Fig 13-15c
Humans
Long Interspersed Elements (LINEs) & SINEs Atypical – not organized like standard retrotransposon Still has pol LINEs are autonomous & SINEs are nonautonomous
T Western Biol 202
Fig 14-20
17
Return to nucleus as RNA with 2 encoded proteins
Transposition of LINEs (L1)
ORF2 = endonuclease to cut DNA + reverse transcriptase to copy RNA into DNA
Cytoplasm
Nucleus T Western Biol 202
18
Eukaryotic transposable elements 2 types:
Class I - Retrotransposons LTR retrotransposons LTR
GAG
POL
LTR
Non-LTR retrotransposons LINEs -polyA SINEs 5’UTR
ORF1
ORF2
3’UTR
-polyA
Class II ‒ DNA transposons TIR
transposase
(ORF2)
TIR
N. Juretic T Western Biol 202
19
Barbara McClintock’s “controlling elements” are DNA transposons
Ds is nonautonomous Requires Ac to move
Ac is autonomous
T Western Biol 202
Fig 14-4
20
Barbara McClintock’s “controlling elements” are DNA transposons Ac/Ds of maize Ds elements are Ac elements with internal deletions that lead to loss of transposase function
Other families of autonomous/ nonautonomous transposable elements in maize E.g. En/Spm
T Western Biol 202
21
Barbara McClintock’s “controlling elements” are DNA transposons Transposition mechanism of Ac All done by transposase
Transposons can leave “footprints” if recombination is imprecise Will become stable mutation rather than return to wildtype condition T Western Biol 202
Fig 14-18
22
Hybrid dysgenesis is the result of DNA transposons (P elements) Crosses of wild isolate males (P) x lab strain females (M) Female (wild)
F1 progeny sterile – chromosome breakage, unstable mutations in germ line Unstable mutations of white from dysgenic cross reminscent of what seen before…
Fig 14-15
Male (lab stock)
T Western Biol 202
23
Hybrid dysgenesis is the result of DNA transposons (P elements) P elements of Drosophila Are also nonautonomous versions with internal deletions
T Western Biol 202
Fig 14-16
24
Hybrid dysgenesis is the result of DNA transposons (P elements) Hybrid dysgenesis explained: P cytotype (wild) strains have a repressor of P element transposition in germline cells M cytotype (lab) strains do not so P can transpose Since mother contributes cytoplasm to zygote, only a problem with M females
T Western Biol 202
Fig 14-17
25
Hybrid dysgenesis is the result of DNA transposons (P elements) P-repressor & Ptransposase encoded by alternative splicing: Somatic cells – intron 3 unspliced repressor Germ cells – all introns spliced transposase
EG 21-24
T Western Biol 202
26
Hybrid dysgenesis is the result of DNA transposons (P elements)
In P cytotype females, Prepressor deposited into oocytes, so “immune”
EG 21-25
T Western Biol 202
27
Eukaryotic transposable elements 2 types:
Class I - Retrotransposons LTR retrotransposons LTR
GAG
POL
LTR
Non-LTR retrotransposons LINEs -polyA SINEs 5’UTR
ORF1
ORF2
3’UTR
-polyA
Class II ‒ DNA transposons TIR
transposase
(ORF2)
TIR
N. Juretic T Western Biol 202
28
Harnessing of eukaryotic transposable elements P elements as a molecular biology tool Insertional mutagenesis Let transposons “jump” & isolate rare stable mutants Mutants created by insertional mutagenesis have a “tag” of known DNA sequence so easy to clone affected genes (often called “transposon tagging”) T Western Biol 202
Fig 14-17
29
Harnessing of eukaryotic transposable elements
P elements for Drosophila transformation
Fig 14-19
Use to “transform” flies with particular genes P element used as carrier to insert a gene into the chromosomes of germ cells of the dysgenic flies Progeny will reflect the expression of that gene T Western Biol 202
30
Transposable elements & host genomes DNA sequences that can move and multiply within the genome selfish DNA
self-replication: necessary and sufficient explanation
Explanation for the C-value Paradox
N. Juretic T Western Biol 202
31
Transposons & genome size C value paradox Genome size doesn’t correlate with complexity of organism or with gene number Organism
Genome size (Mbp)
Number predicted genes
Yeast
12
6,144
Arabidopsis
125
25,706
Rice
400
37,500
Maize
2500
50,000
C. elegans
103
20,598
Drosophila
170
13,525
Pufferfish
329
22,089
Humans
3223
~24,000 T Western Biol 202
32
Transposons & genome size Larger genome, more transposons… Organism
Genome size (Mbp)
Number predicted genes
Percent genome is transposons
Yeast
12
6,144
Arabidopsis
125
25,706
10.5%
Rice
400
37,500
35%
Maize
2500
50,000
80%
C. elegans
103
20,598
6.5%
Drosophila
170
13,525
3.1%
Pufferfish
329
22,089
2.7%
Humans
3223
~24,000
45% T Western Biol 202
33
Transposons & genome size Larger genome, more transposons…
Fig 14-22 T Western Biol 202
34
Transposons & the human genome ~45% of genome is transposable elements Figs 14-20
Fig 14-21
T Western Biol 202
35
Transposable elements & host genomes DNA sequences that can move and multiply within the genome selfish DNA
self-replication: necessary and sufficient explanation
How do these selfish elements avoid killing their hosts? If host dies or goes extinct, no transposable elements
N. Juretic T Western Biol 202
36
How can so many transposons be maintained? Fig 14-15
Transposon graveyard
Female (wild)
Male (labstock)
most are “dead” Lost transposase or mutations to flanking repeats
Inactive transposons Kept off by transposon repressors Tend to activate under stress conditions T Western Biol 202
37
How can so many transposons be maintained? Inconspicuous Found in between genes & in introns
Fig 14-21
T Western Biol 202
38
How can so many transposons be maintained? Inconspicuous Found in between genes & in introns Insert into each other (looks like just one to genome…)
Nearly all RTE insertions are into even older RTEs. A portion of a maize chromosome is illustrated. (Walbot et al 2001 PNAS) T Western Biol 202
39
How can so many transposons be maintained? Inconspicuous Found in between genes & in introns Insert into each other (looks like just one to genome…) Safe havens
T Western Biol 202
40 Fig 14-23b
How can so many transposons be maintained? Host is able to inactivate Often found in heterochromatin – kept off by methylation, etc Other host mechanisms related to those used to suppress virus replication
Fig 13-29 T Western Biol 202
41
Transposable elements & host genome evolution DNA sequences that can move and multiply within the genome selfish DNA
self-replication: necessary and sufficient explanation
Transposable elements can be harnessed by their hosts for genomic elements & genome evolution
N. Juretic T Western Biol 202
42
Transposable elements & genome structure Useful Structural role around centromeres? Telomeres of Drosophila - composed of retrotransposons – HeT-A & TART
T Western Biol 202
43
Transposable elements & genome structure Mediate genome evolution? New combinations of genes or gene elements through recombination between homologous transposable elements
T Western Biol 202
44
