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Lecture 17: !

Transposable Elements - Jumping Genes"

2"

Transposable elements are mobile (able to transport themselves)


genome sequences (internal not external elements e.g. virus).
Transposable elements or transposons are put in 3 categories that
differ in transposition mode, mechanism and location in the host:
(a) Cut &paste transposase enzymes are used to cut (excise from a
location) and paste (insert) into a new genomic site.
(b) Retrotransposons use reverse transcriptase to copy template RNA
into DNA and insert it with integrase (in host genomic DNA).
(c) Replicative transposons: a mobile element in an extra
chromosomal donor plasmid uses transposase to catalyze the cointegration (fusion) with a recipient plasmid, copying the
transposon into the recipient.
Focus: category definition, mechanism and identification

3"

Transposable elements are mobile elements of DNA


that can change position in the genome, insert into
genes and may cause mutations if they insert in a
coding region.

Transposable elements were discovered by


Barbara McClintock for which she received the Noble
Prize.

C. R. Burnham Marcus Rhodes Rollins Emerson

1929
Cornell University
Ithaca, New York

George Beadle

Pudgie

4"

female gametophyte: egg cell and 2 polar


nuclei; !
male gametophyte: 3 nuclei."
Double fertilization:"
a) 1 sperm nucleus and egg nucleus=
embryo!
b) 1 sperm nucleus and 2 polar cells =
triploid endosperm!

B. Mclintock
discovered
transposable
elements while
studying
unstable genes
that regulate the
color of the
aleurone layer!

Outer layer of the


endosperm forms the
aleurone layer!

C dominant allele inhibits aleurone color!


Bz dominant purple aleurone color!
Ds dissociator gene chromosome breakage, !
Wx- produces starch stain purple with iodine, Sh - plump!
"
cytological!
2 strains: !
" marker!
female homozygous,
all recessive (2 copies
but show one), !
male hemizygous
dominant !

c - recessive allele produces aleurone color (any kind)"


bz recessive bronze (dark brown) color "
wx waxy or stains brown with iodine, sh - shrunken!

Mapping studies indicated the Ds (dissociation factor) was


related to chromosome breakage. An unlinked factor Ac
(activator) was required to activate breakage."

(1) but it was impossible to map Ac to a consistent locus!


"
5"

Experiment ( a) Ds is a genetic element that may cause


chromosomal breakage.!
"
Experiment (b). Ds can move!

Transposition and color: using linked dominant markers!

Figure 14-3"

a) Lacking a knob and!


most were colorless,
shrunken, waxy . "
Breakage between wx and
the centromere, !
Ds mapped , 1cM from wx!

"

6"

b) Color mosiacs: Ds moves


insertion inactivates C . Sh &
Wx expression was normal (or
between C & bz) . Mosaic
happens in different cell lines!

Experiment (c) Ac controls Ds breakage and can move.


And the effect is heritable."
"
Experiment (d) Ds can insert into a gene. When it does, it
produces an unstable gene."

c) Ds moves Ds maps to C, !
Color is blocked Ds inserts
into C gene, unless Ac is
present (sometimes
mapped), it stays."
d) Unstable : Ds transposes"
Color is expressed in some"
aleurone cells. "
But same for starchy/ waxy in
unstable color lineDs moves
and leaves a copy behind!

"

But Ac mapped to the same


location in unstable alleles.
( same for waxy)!
Ac and DS related ? !
Now Known

Ac - autonomous elements - self mobile!


Ds non-autonomous - require other elements for mobility! 7"

N. Federoff, S. Wessler and M. Shure.1983.Cell 35 "


They cloned restriction endonuclease fragments containing
part of the waxy locus, also,!
insertion mutants with Ac and Ds found..!
Also!
1) Ac element is almost exactly identical to the Ds element
(measured as same restriction sites) ."
2) But Ac and Ds elements are not internally repetitive, and are
inserted in opposite orientations."
3) Sequences homologous to Ac termini are more abundant
than are sequences homologous to the center of the element.!

Class 2:EUKARYOTIC CUT AND PASTE TRANSPOSONS"

DNA transposons in the Ac (Ds) family


move because of an!
Ac encoded, trans-acting transposase,
an enzyme that gets into the
nucleus, and binds to specific
terminal sequences (11 nucleotides,
inverted repeats)!
causing:!
non-replicative excision and !
transposition, causing insertions and
deletions, sometimes chromosome
breakage and translocations.!
Definition, mechanism,identification

8"

8"

Ac elements are 4563 base pairs bounded by inverted terminal


repeats (11 nucleotides), which are necessary for transposition
through this family's transposase."
!
Terminal invered repeat sequence defines a transposon family. !
Ds has the same terminal repeats as Ac!
!
ATCGCA..transposase..TGCGAT!
TAGCGT ....ACGCTA!
!
But Ds element internal sequences vary in additions, deletions and
insertions (translocation and transposition) "
(1) internal deletions leaving the transposase enzyme non-functional!
(2) they may contain non-Ac DNA (aberrant Ac elements);!
(3) one Ds element may be inserted into another (double DS
elements). These cause chromosome breakage.!
!
9"

An inserted element is flanked by a short direct repeat"


When Ac inserts with a
staggered cut it marks the site
with 8 nucleotide pairs.
These direct repeats - target
site duplications, are not
part of the Ac element. "
"
Direct repeats (target site
duplications) mark an
insertion site when the
staggered cut is repaired.!
!
The repair to the staggered
cut makes (inserts) direct
repeats due to the staggered
cut and are 2 fold rotational
symmetric due to the repair
of the staggered cut.!
10"

McClintocks results were largely ignored for 20


years, until the first transposable elements were
isolated from bacterial DNA in the 1960s. "
"
The first eukaryotic DNA transposons that were
characterized at the molecular level were the"
P elements in Drosophila, first identified more than
17 years later in 1977-80 . "
"
However, Barbara Mclintock went from one major
piece of work to new discoveries beginning with the
first physical demonstration of chromosome
recombination, cytology and chromosome genetics
and genome organization leading to her Nobel Prize. "
11"

EUKARYOTIC CUT AND PASTE TRANSPOSONS"


A different cut and paste family of transposons was discovered in
Drosophila,
Terminal sequence for binding

"

P element inverted
repeats are 32
nucleotides long,
specific for each
family and when
they insert they
initiate a repair
8bp inverted
repeats long.!
12"

P strain and M strain Drosophila first appeared inAustralian


fly stocks in the 1960's

Flies from strains captured before 1950 do not have P elements, wild
populations vary in the number of P elements, some close relatives of Drosophila
melanogaster have no P elements , distant fly relatives have some."
P elements were reported in in fly stocks during the 1960's. They were not
observed in any fly stocks isolated before 1950. "
What made them noticeable was that two mating type strains were first seen
over a 10 15 year period in recently isolated fly strains and in lab strains
exchanged between Australia and United States and used in both countries.
Crosses between two strains produced dysgenic M-P hybrids that had
frequent mutations, chromosome breakage and sterility. One strain named the P
for paternal P and M for maternal. Populations that possess P elements
have mechanisms to regulate their movement, more specifically, a repressor
in a P cytotype represses P element movement. The M cytotype permits it. This
repressor appear to target mRNA for destruction by a small RNAs - RNA
interference. "

Insertion of P elements cause mutations underlying hybrid dysgenesis"

female"
M"
P"
male"

M"
P"

"

normal" normal
normal"
dysgenic!
dysgenic" normal"

13"

Another kind of eukaryotic transposons"


Class 1- retrotransposons!

Retrotransposons, inserted elements that


resemble retroviruses, a single stranded RNA
that uses double stranded DNA as an
intermediate.. "
14"

Life cycle of a retrovirus"


A retrovirus is a
single stranded
RNA molecule
that uses double
stranded DNA as
an intermediate"

Figure 14-11"

15"

16"

A retrotransposon is
transposed through an
RNA intermediate, using
reverse transcriptase/
integrase in pol. "
gag encodes a structural
protein of the virus-like
capsule. "
LTR retrotransposons "
are flanked with "
Long Terminal Repeats. !

Retrotransposons have features in common with retroviruses"


LINES"
- Long Interspersed
Element: repeated
sequence(S),
Satellite DNA
(1-7,000bp)
interspersed with
unique sequence:"
Promoter, and !
2 open reading frames
ORF:!
(1)nucleic acid binding
protein (gag).!
(2)endonuclease and
reverse transcriptase
(pol).!

17"

DR- DIRECT REPEAT, IR INVERTED REPEAT, LTR LONG TERMINAL


REPEAT,TSD TARGET SITE DUPLICATION

http://www.nature.com/
scitable/topicpage/
transposons-the-jumpinggenes-518

Rt reverse transcriptase, en- endonuclease Gag group specific


antigen, prt protease, env envelope protein, TSD target site
duplication,DR direct repeat, ITR inverted terminal repeat

Organisms with smaller genomes have less transposable


element sequence or genome sizes correlates with the amount
of DNA in the genome that is derived from transposable
elements!
What is the prevalence of transposable elements in the human
genome?

Non-retroviral like!

18"

19

Eukaryotes
Structure!

Genes
Encoded!

Transposition!

Examples!

Cut &Paste!

Short terminal
Transposase
inverted repeats
gene!
characteristic of a
family. Target site
direct repeats!

Cut and paste,


AcDs family
excise and insert in maize!
DNA!
P element in
Drosophila!

Retrotransposons!

Long terminal
direct repeats.
Target site (short)
direct flanking
insertion repeats!

Through an
RNA
intermediate
using an
integrase to open
host DNA,
reverse
transcriptase to
insert DNA!

Reverse
transcriptase,
integrase (pol)
and a protein
(gag)!

Ty1 (yeast)!
Copia
(Drosophila)!
LINES , L1
or Alu in
humans!

20"

Transposition in Prokaryotes"

(1)Bacterial Insertion sequences, (IS) may encode a transposase (cut & paste)
only, they can move around the bacterial genome, and insertions are often
deleterious. These transposons also have characteristic, inverted terminal
repeats and direct target site duplications.

"

(2) Composite A variety of


genes may reside between 2
(nearly identical, different
directed), IS elements If both
IS contain an active
transposase the entire
sequence between them is
captured and can jump. "

(3) Simple transposons (Tn3) do not have IS elements,


they are larger than IS sequences, but they have a
transposase enzyme and they may have incorporated
other DNA. "

"

21"

http://
www.sci.sdsu.edu/
~smaloy/
MicrobialGenetics/
topics/transposons

Replicative
transposition of
Tn3"

http://www.sci.sdsu.edu/
~smaloy/MicrobialGenetics/
topics/transposons

Replicative Transposons They are


on plasmids- extrachrosomal,
circular DNA."
They transpose by a replicative
mechanism leaving a copy on the
donor and recipient plasmid or
chromosome (Fig 14-10). The
transposase in Tn3 catalyzes the
formation of a co-integrate
between the donar and recipient
plasmid (cointegrate) . The
transposon is replicated in the
process. The resolvase mediates
recombination between the 2 Tn3
transposons leaving a copy on both
plasmids."

Prokaryotes" Structure!

Genes
Encoded!

Transposition!

Examples!

IS elements!
!
!
!
!
"
Composite"
"
transposons!

Short terminal
Transposase
inverted repeats gene!
characteristic of
a family. Target
site direct
repeats!
!
Flanking IS
elements may be
in an inverted or
repeated
orientation!

Cut and paste,


excise and insert
DNA!

IS1, IS2, IS10!


!
!
!
!
!
!
Tn10!

"

No IS elements!
Characteristic
terminal
inverted repeat
sequences!

!
Replicative
mechanism!

!
Tn3!

Simple
transposons!

!
Transposase
gene &
Resolvase!

A composite R plasmid may contain several transposons carrying


resistance genes. If the plasmid and bacterial chromosome recombine at a common
IS site, it will be integrated into the bacterial chromosome, and can be
transferred across species during congugation."
The spread of multiple
drug resistance is
accelerated by
congugative R
plasmids containing:"
"
(1) Resistance transfer
factor (F+ genes for
congugation)"
"
(2) R determinant (has
genes for antibiotic
resistance flanked by 2
insertion (IS)
sequences).!
"
"

23"

24"

Bacterial Transposon sequence can move between the


bacterial chromosome and plasmids (small circular extrachromosomal DNA), one plasmid to another or between
bacterial genomes, but the transposition rate of composite
transposons is very low. How does this mechanism become the
major way of generating multiply-resistant superbugs ?"
"

Bacteria get other bacterial DNA by several


processes!
Transformation!
Plasmid donation!
(Partial) genome donation!
Transduction!
"

Any piece of
bacterial
genome or
plasmid could
have a
transposon"

25"

F plasmids have a 1 way transfer during conjugation,


from F+ to F- cells. F+ plasmids have genes for
congugation"

26"

Integration of the F plasmid creates an Hfr (High


frequency recombination) strain"
The F plasmid has 2 IS
elements (IS2,3). "
"
When there are IS
elements in a plasmid
and a chromosome,
different DNA molecules
(Bacterial chromosome &
plasmid) may recombine,
forming a High frequency
recombination (Hfr) strain
that makes up to 1000
times as many
recombinants as F+
strains"
27"

Multiple resistance R- Plasmids from chapter 5."


A plasmid with segments from many former bacterial
hosts"

23"

Table 5-3 Molecular Biology of the Cell ( Garland Science 2008)"